Composition and method for the prevention and treatment of obesity

ABSTRACT

A method for reducing the weight of an overweight or obese subject comprising administering a therapeutically effective amount of a composition comprising  64 Zn-enriched zinc. A method for preventing or reducing weight gain comprising administering an effective amount of a composition comprising  64 Zn-enriched zinc.

TECHNICAL FIELD

This disclosure relates to prevention and therapy for overweightness andobesity.

BACKGROUND

Obesity is currently a chronic metabolic disease that can occur at anyage and is characterized by an excessive increase in body weight mainlydue to excessive accumulation of adipose tissue. Overweightness andobesity are the most common nutrition-related problems in developedcountries. According to data from WHO, more than 1.9 billion adults aged18 years and older are overweight. Of these, over 600 million are obese(World Heath Organization, 2015). The number of newly diagnosed cases ofthese disorders increases every year. More worrying is that the levelsof childhood obesity are increasing at alarming rates. Thus, in 2014,about 40 million children under the age of 5 were overweight. Such asituation necessarily causes concern, given that childhood obesity is aserious predictor of obesity in adulthood and significantly increasesthe risk of premature death or disability.

Currently pharmacotherapy for obesity is limited, ineffective, and/orwith side-effects.

SUMMARY

In one aspect, this disclosure provides a composition comprising zincthat is ⁶⁴Zn-enriched zinc (the term “⁶⁴Zn_(e)” is used herein to referto ⁶⁴Zn-enriched zinc); the composition is provided at a therapeuticallyor prophylactically effective amount (dose) for preventing or treatingobesity and overweightness. In another aspect, a method of use of saidcomposition is provided. In some embodiments, the ⁶⁴Zn-enriched zinc isin the form of a ⁶⁴Zn_(e) compound or a ⁶⁴Zn_(e) salt.

The disclosed composition contains zinc that is enriched for ⁶⁴Zn. Incertain embodiments, the disclosed compositions contain zinc that is atleast 80% ⁶⁴Zn_(e), at least 90% ⁶⁴Zn_(e), at least 95% ⁶⁴Zn_(e), or atleast 99% ⁶⁴Zn_(e), for example, zinc that is 80% ⁶⁴Zn_(e), 85%⁶⁴Zn_(e), 90% ⁶⁴Zn_(e), 95% ⁶⁴Zn_(e), 99% ⁶⁴Zn_(e), or 99.9% ⁶⁴Zn_(e).

In another aspect, the disclosure provides a method of administering aprophylactically or therapeutically effective amount of a disclosedcomposition to a subject to decrease the subject's weight or to preventor decrease weight gain. In some embodiments, the subject may beoverweight or obese. In some embodiments, the subject may be someone whowishes to avoid becoming overweight or obese. The subject may be a humanor a non-human mammal, such as a non-human primate or a domesticated dogor cat.

Numerous other aspects are provided in accordance with these and otheraspects of the invention. Other features and aspects of the presentinvention will become more fully apparent from the following detaileddescription and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the dynamics of body weight gain for animals inexperimental groups (M±n, n=10).

FIG. 2 shows the calorie content of food consumed by animals of theexperimental groups (M±n, n=10).

-   Note: 1—Control; 2—Control+⁶⁴Zn_(e) stable isotope in aspartate    form; 3—diet induced obesity+⁶⁴Zn_(e) stable isotope in aspartate    form; 4—Diet Induced Obesity.

FIG. 3 shows insulin levels in the blood of experimental animals (M±n,n=10).

FIG. 4 shows pancreatic islet area of experimental animals (M±n, n=10).

FIG. 5 shows Dynamics of increase in body weight of animals inexperimental groups (M±n, n=10).

-   Note: C—control; C+zinc—control on the background of administration    of Zn-64 stable isotope in aspartate form; DIO—diet induced obesity;    DIO+zinc—diet induced obesity on the background of administration of    Zn-64 stable isotope in aspartate form.

FIG. 6 shows Caloric content of food consumed by animals of experimentalgroups (M±n, n=10).

-   Note: 1—Control; 2—Control+Zn-64 stable isotope in aspartate form;    3—Obesity+Zn-64 stable isotope in aspartate form; 4—Obesity.

FIG. 7A-FIG. 7F show micrographs of sections of pancreas in animals fromthe control (FIG. 7A-FIG. 7C) and obesity (FIG. 7D-FIG. 7F) groups,hematoxylin & eosin, arrows show exocrine cells with marked fattydegeneration, eye. 10× obj. 10, eye. 10× obj. 40.

FIG. 8A-FIG. 8F show micrographs of sections of pancreas in animals fromthe control group (FIG. 8A-FIG. 8C) treated with Zn-64 stable isotope inaspartate form and animals from the obesity group (FIG. 8D-FIG. 8F)treated with Zn-64 stable isotope in aspartate form, hematoxylin &eosin, eye. 10× obj. 10, eye. 10× obj. 40.

FIG. 9 shows cross-sectional surface area of the islets of Langerhans.

-   *—the difference between the control and experimental groups is    significant when p≤0.05; #—the difference between the obesity group    and obesity group treated with Zn-64 stable isotope in aspartate    form is significant when p≤0.05.

FIG. 10A-FIG. 10D show micrographs of sections of liver in animals fromthe control (FIG. 10A and FIG. 10B) and obesity (FIG. 10C and FIG. 10D)groups, hematoxylin & eosin, eye. 10× obj. 10, eye. 10× obj. 40.

FIG. 11A-FIG. 11D show micrographs of sections of liver in animals fromthe control (FIG. 11A and FIG. 11B) and obesity (FIG. 11C and FIG. 11D)groups, all animals treated with Zn-64 stable isotope in aspartate form,hematoxylin & eosin, eye. 10× obj. 10, eye. 10× obj. 40.

FIG. 12A (hepatocyte nucleus area), FIG. 12B (hepatocyte area), and FIG.12C (nucleus-to-cytoplasm ratio of hepatocytes) show morphologicalanalysis of the liver.

-   *—the difference between the control and experimental groups is    significant when p≤0.05; #—the difference between the obesity group    and obesity group treated with Zn-64 stable isotope in aspartate    form is significant when p≤0.05.

FIG. 13A-FIG. 13D show micrographs of sections of liver in animals fromthe control (FIG. 13A and FIG. 13B) and obesity (FIG. 13C and FIG. 13D)groups, Van Gieson's staining method for the detection of collagenfibers (fibrosis) eye. 10× obj. 10, eye. 10× obj. 40.

FIG. 14A-FIG. 14D show micrographs of sections of liver in animals fromthe control (FIG. 14A and FIG. 14B) and obesity (FIG. 14C and FIG. 14D)groups, all animals treated with Zn-64 stable isotope in aspartate form,Van Gieson's stainin method for the detection of collagen fibers(fibrosis), eye. 10× obj. 10, eye. 10× obj. 40.

FIG. 15 shows morphometric analysis of liver fibrosis. *—the differencebetween the control and experimental groups is significant when p≤0.05;#—the difference between the obesity group and obesity group treatedwith Zn-64 stable isotope in aspartate form is significant when p≤0.05.

FIG. 16A-FIG. 16E show analysis of body weight, food and waterconsumption by experimental groups of animals. FIG. 16A Food consumptionrates in grams per animal; FIG. 16B Water consumption rates in ml per 1animal; FIG. 16C Average daily food consumption (during the experiment)per 1 animal; FIG. 16D Average daily water consumption (during theexperiment) per 1 animal; FIG. 16E Weight gain in rats 2 weeks afterdrug administration.

FIG. 17 is a graph showing serum insulin level (CU/mg of total protein).

FIG. 18A (serum) and FIG. 18B (liver) are graphs showing superoxidedismutase activity (antioxidant Zn-dependent enzyme) CU/mg*min.

FIG. 19 is a graph showing measurement of the area of islets ofLangerhans in the pancreas of laboratory animals (microscopically, atday 7 after the last administration of drugs).

FIG. 20A-FIG. 20F are microscopic photos of the of islets of Langerhans.FIG. 20A and FIG. 20B—top panel; FIG. 20C and FIG. 20D—middle panel;FIG. 20E and FIG. 20F—bottom panel.

FIG. 21A-FIG. 21C shows accumulation of metals in liver tissues oflaboratory animals.

FIG. 22A-FIG. 22C shows accumulation of metals in kidney tissues oflaboratory animals.

FIG. 23A-FIG. 23F are microscopic photos of the of islets of Langerhans,FIGS. 23A and 23B—control group, magnification ×10 and ×40,respectively, FIGS. 23C and 23D—zinc acetate, comparison group,magnification ×10 and ×40, respectively, FIGS. 23E and 23F—zinc isotope,therapeutic group, magnification ×10 and ×40, respectively.

DETAILED DESCRIPTION

As used herein, the word “a” or “plurality” before a noun represents oneor more of the particular noun.

For the terms “for example” and “such as,” and grammatical equivalencesthereof, the phrase “and without limitation” is understood to followunless explicitly stated otherwise. As used herein, the term “about” ismeant to account for variations due to experimental error. Allmeasurements reported herein are understood to be modified by the term“about,” whether or not the term is explicitly used, unless explicitlystated otherwise. As used herein, the singular forms “a,” “an,” and“the” include plural referents unless the context clearly dictatesotherwise.

“Effective amount,” “prophylactically effective amount,” or“therapeutically effective amount” refers to an amount of an agent orcomposition that provides a beneficial effect or favorable result to asubject, or alternatively, an amount of an agent or composition thatexhibits the desired in vivo or in vitro activity. “Effective amount,”“prophylactically effective amount,” or “therapeutically effectiveamount” refers to an amount of an agent or composition that provides thedesired biological, therapeutic, and/or prophylactic result. That resultcan be reduction, amelioration, palliation, lessening, delaying, and/oralleviation of one or more of the signs, symptoms, or causes of adisease, disorder or condition in a patient/subject, or any otherdesired alteration of a biological system. An effective amount can beadministered in one or more administrations.

An “effective amount,” “prophylactically effective amount,” or“therapeutically effective amount” may be first estimated either inaccordance with cell culture assays or using animal models, typicallymice, rats, guinea pigs, rabbits, dogs or pigs. An animal model may beused to determine an appropriate concentration range and route ofadministration. Such information can then be used to determineappropriate doses and routes of administration for humans. Whencalculating a human equivalent dose, a conversion table such as thatprovided in Guidance for Industry: Estimating the Maximum Safe StartingDose in Initial Clinical Trials for Therapeutics in Adult HealthyVolunteers (U.S. Department of Health and Human Services, Food and DrugAdministration, Center for Drug Evaluation and Research (CDER), July2005) may be used. The person of ordinary skill in the art is aware ofadditional guidance that may also be used to develop human therapeuticdosages based on non-human data. An effective dose is generally 0.01mg/kg to 2000 mg/kg of an active agent, preferably 0.05 mg/kg to 500mg/kg of an active agent. An exact effective dose will depend on theseverity of the disease, patient's general state of health, age, bodyweight and sex, nutrition, time and frequency of administration,combination(s) of medicines, response sensitivity and tolerance/responseto administration and other factors that will be taken into account by aperson skilled in the art when determining the dosage and route ofadministration for a particular patient based on his/her knowledge ofthe art. Such dose may be determined by conducting routine experimentsand at the physician's discretion. Effective doses will also varydepending on the possibility of their combined use with othertherapeutic procedures, such as the use of other agents.

As used herein, a “patient” and a “subject” are interchangeable termsand may refer to a human patient/subject, a dog, a cat, a non-humanprimate, etc.

The terms “overweight” (or “overweightness”) and “obesity” refer to bodyweight that is greater than what is considered normal or healthy for acertain height. A person's body mass index (BMI) is one way to tell ifone is at a healthy weight, overweight, or have obesity. The BMI is ameasure based on a person's weight in relation to height. The greaterthe BMI, the greater risk of health problems from overweight andobesity. Overweight may be defined as having a BMI between 25 and 29.9.Obesity may be defined as having a BMI of 30 or more.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Obesity

Obesity is a chronic metabolic disease that can occur at any age and ischaracterized by an excessive increase in body weight mainly due toexcessive accumulation of adipose tissue. Overweightness and obesity arethe most common nutrition-related problems in developed countries.According to data from WHO, more than 1.9 billion adults aged 18 yearsand older are overweight. Of these, over 600 million are obese (WorldHeath Organization, 2015). The number of newly diagnosed cases of thisdisease increases every year. More worrying is that the levels ofchildhood obesity are increasing at alarming rates. Thus, in 2014, about40 million children under the age of 5 were overweight. Such a situationnecessarily causes concern, given that childhood obesity is a seriouspredictor of obesity in adulthood and significantly increases the riskof premature death or disability.

Thus, the growing dynamics of the prevalence of this disease amongdifferent segments of population, regardless of social and professionalaffiliation, place of residence, age or gender, brings the problem ofobesity to the level of socially important issues, the solution of whichrequires immediate action and innovative approaches to the preventionand treatment of this pathology.

Obesity develops as a result of disruption of normal metabolism, theprocess of converting food to energy on a cellular level. Obesity is amultifactorial disease, the etiology and pathogenesis of which isassociated with the cumulative effect of many factors and is accompaniedby a disruption in the functioning of most body systems. Overweightnessand obesity are the main risk factors for the development of a number ofco-morbidities and complications, including, for example, cardiovasculardiseases, hypertension, diabetes mellitus type 2, certain cancers,dyslipidemia, fatty liver disease and cirrhosis, osteoarthritis andvarious mental disorders. Obesity is one of the leading preventablecauses of death worldwide, with increasing rates in adults and children,and is considered as one of the most serious public health problems ofthe 21st century.

A long-term activation of oxidative stress resulting from excessivereactive oxygen species (ROS) production and intensification of freeradical reactions occurring against the background of depletion of thebody's antioxidant reserve is one of the key pathogenetic factorsunderlying the pathogenesis of both obesity and obesity-relateddiseases. Another mechanism involved in the development and progressionof obesity-related disorders is associated with the induction of achronic inflammatory process. Its low intensity does not give directclinical symptoms, but at the same time, this process is systemicbecause it covers a wide range of organs and tissues which leads tochanges in their functional activities.

Given the multifactorial nature of obesity, its treatment should becomprehensive and based on a stepwise strategy that includes the use ofnon-drug approaches (diet therapy, exercise, correction of behavior anddietary pattern) along with the use of certain therapeutic agents and,in extreme cases, surgical intervention.

Existing therapeutic anti-obesity agents are divided into broad classesof appetite suppressants, fat absorption inhibitors and stimulants thathelp increase calorie burn. Pharmacological agents that are currentlywidely used for the treatment of this pathology have a number of seriousside effects. When administered for a long period or used as amonotherapy, they are able to reduce body weight by no more than 8 to10% per year. Anti-obesity drugs that are currently approved forlong-term use include orlistat (trade name: XENICAL) and sibutramine(trade name: REDUCTIL) (Haslam, D. “Weight management in obesity—pastand present.” Int J Clin Pract. 2016:206-17). Orlistat, as a lipaseinhibitor, inhibits pancreatic lipase reducing digestion and absorptionof fat from the small intestine, and sibutramine, as an appetitesuppressant, inhibits norepinephrine and serotonin reuptake. However,orlistat induces malabsorption of fat-soluble vitamins, abdominaldiscomfort, bloating, oily stool, and the like, and sibutramine causesside effects such as headache, dry mouth (heavy thirst), insomnia,constipation, and the like.

Thus, the pharmacotherapy for obesity is limited and associated withside-effects. Anti-obesity medications, as a rule, provide onlyshort-term improvement, but not lasting, long-term effect. Therefore,there is a huge unmet need for effective anti-obesity agents that wouldnot have side effects and would be safe when used for an extended periodof time.

A variety of methods and means of the prior art are based on the use ofdiets high in fiber, vitamins and other biologically active components(cereals and whole grains, vegetables, fruits, nuts, greens, etc.) andlow in carbohydrates that are easily absorbed by the body (sugar,sweets, pastries, bakery products and pasta made of high-grade flour)(patent RU2147228, publ. date: Apr. 10, 2000), as well as on physicalexercise. However, diet changes are effective in conducting clinicaltrials, but not so effective in real life because of difficulties withkeeping a diet on a daily basis. In addition, a number of scientistsbelieve that diets are useless for the treatment of obesity.

There is a composition for the prevention and treatment of obesitydescribed in the art that comprises a moneywort extract (Lysimachiaefoenum-graeci herba) as an active ingredient (RU2475256, publ. date:Feb. 20, 2013). The said extract has a low cytotoxicity but it inhibitsdifferentiation of adipocytes (fat cells) and leads to a decrease in thebody weight and body fat in animal models of obesity. However, the saidextract does not provide sufficient effectiveness.

Another known anti-obesity agent is a combination of a-lipoic acid andN-acetylcysteine (RU2670612, publ. date: Aug. 17, 2018). α-lipoic acidis a microcomponent that can be found in some foods such as yeast, andorgan meats such as liver. α-lipoic acid physiologically acts as acoenzyme closely related to thiamine in a complete oxidativedecarboxylation system of pyruvic acid or alpha-ketoglutaric acid, andalso functions as an antioxidant. N-acetylcysteine interacts directlywith peroxide, peroxide-hydrogen, hydroxyl radical, reducing oxidativestress, or has an antioxidant effect by providing cysteine, which is thestarting material in glutathione biosynthesis, indirectly increasingglutathione synthesis. However, α-lipoic acid can induce such adverseeffects as dyspepsia (heartburn, abdominal pain, nausea, diarrhea),hypoglycemia (decrease in blood sugar levels), and allergic reactions,and N-acetylcysteine can provoke an increase in systolic and diastolicpressure in elderly people.

WO2018144911 (publ. date: Aug. 9, 2018) describes a weight losscomposition that includes protein, vitamins A, C, E, zinc and magnesium.The composition has a low calorie content, but elemental zinc is verypoorly absorbed in the body, which reduces the effectiveness of suchcomposition for the treatment of obesity. The said composition canrather be used as a dietary supplement.

Another method for body weight management is described in WO2016193067(publ. date: May 10, 2018). It involves the use of a compositioncomprising cinnamaldehyde and a zinc salt selected from the groupconsisting of zinc sulfate, zinc lactate and zinc citrate, whereincinnamaldehyde:zinc ratio is preferably 1:0.5 to 1:0.005, morepreferably 1:0.03 (in molarity)”. The said composition is recommended toimprove insulin sensitivity, glucose tolerance, cognitive performance,cognition, mood and memory. This composition also provides for the useof zinc salts, which does not make it possible to achieve high efficacydue to the low bioavailability of this element.

Zinc

Zinc is attributed to the trace elements which are essential forensuring a proper metabolic status of the human body. More than 200enzymes throughout the body depend on zinc. This element is either aconstituent of enzymes or a regulator of their activity covering allclasses of enzymes: transferases (RNA and DNA polymerases, reversetranscriptase, thymidine kinase, nucleotidyl transferase,carboxypeptidase and other peptidases), hydrolases (alkalinephosphatase, 5-nucleotidase, aminopeptidase, etc.), lyases (aldolase,carbonic anhydrase, etc.), oxidoreductases (alcohol dehydrogenase,superoxide dismutase, etc.), ligases and isomerases. Without zinc, noprotein, fat or carbohydrates metabolism is possible.

Zinc has also been proven to exhibit a mediated antioxidant effect. Zincis an inhibitor of NADPH oxidase, an enzyme complex that catalyzes theproduction of highly aggressive superoxide anion radicals. In addition,it can have a direct effect on the oxidation of free radicals at thestage of initiation of chain reactions; it is a structural component ofsome enzymes of the antioxidant defense system, includingCu/Zn-containing superoxide dismutase. By joining the thiol groups ofproteins, zinc protects them from oxidation by reactive oxygen species.This trace element induces the synthesis of metallothioneins,cysteine-rich proteins acting as free radical scavengers. Zincsuppresses the formation of reactive mixed valence metal oxides and isinvolved in stabilization of the membrane structure.

The metabolic and structural significance of zinc is determined by abroad spectrum of its biological activity. Thus zinc is necessary forthe normal running of processes associated with cell division anddifferentiation (growth, tissue regeneration, spermatogenesis, andothers), and is actively involved in metabolism of nucleic acids andprotein synthesis. This trace element is important for metabolism ofpolyunsaturated fatty acids and reactions of prostaglandintransformations. It shows pronounced lipotropic activity and hashepatoprotective properties. Haase H., Rink L. Zinc Signaling. Zinc inHuman Health//Amsterdam, Netherlands: IOS Press. 2011. 243.

In addition, zinc plays an extremely important role in immunologicalreactions as it is a regulator of the activity of phagocytes andlymphocytes and has an effect on chemotaxis of neutrophils.5-nucleotidase, a zinc-containing enzyme, is of great importance in thefunctional state of T- and B-lymphocytes. Isolated zinc deficiencycauses severe disturbances in various parameters of T-cell function,including thymus involution, inhibition of cell-mediated cytotoxicityand reduction in the total number of lymphocytes. Zinc is involved inmetabolism and stimulation of the activity of pituitary hormones,adrenal glands, pancreas, prostate glands and testes. Zinc plays a clearrole in the synthesis, storage and secretion of insulin. Haase H., RinkL. Zinc Signaling. Zinc in Human Health//Amsterdam, Netherlands: IOSPress. 2011. 243.

Zinc also acts as a synergist/antagonist to absorption of many traceelements and vitamins (iron, copper, magnesium, vitamins A, E, folicacid, and others) and has an effect on their metabolism.

In sum, zinc is involved in a variety of vital processes and functionsin the human body. A detailed study of some of these functions is notyet fully completed, and many of the mechanisms of action of this traceelement are still not fully understood or recognized. However,experimental and clinical studies presented in the literature show zincas one of the key elements, the decrease in the levels of which in thebody is associated with the onset and progression of a number of themost widespread non-epidemic diseases. Since the main metabolicprocesses in the body occur with the active participation ofzinc-containing and zinc-dependent enzymes, its deficiency causes aviolation of many vital processes.

The use of classical pharmacological forms of zinc—zinc salts and itschelates—does not always make it possible to achieve a proper effect ofcompensating for zinc deficiency due to the low bioavailability of thiselement.

Treatments Methods and Compositions

In one aspect, this disclosure provides a composition comprising zincthat is ⁶⁴Zn-enriched zinc at a therapeutically or prophylacticallyeffective amount (dose) for preventing and/or treating obesity andoverweightness. In some embodiments, ⁶⁴Zn-enriched zinc is in the formof a ⁶⁴Zn_(e) compound or a ⁶⁴Zn_(e) salt. In some embodiments, thedisclosed composition comprises ⁶⁴Zn_(e) is in a form of salt selectedfrom the group consisting of aspartate (chemical formula—(C₄H₅O₄N)₂⁶⁴Zn_(e)) with 2 aspartic acid molecules, sulfate, and citrate.

The term “⁶⁴Zn_(e)” is used herein to refer to ⁶⁴Zn-enriched zinc. Thatis, zinc that is enriched for ⁶⁴Zn such that ⁶⁴Zn is enriched greaterthan its usual percentage in zinc in nature.

The disclosed compositions contain zinc that is enriched for ⁶⁴Zn_(e).Zinc in the form of the light isotope ⁶⁴Zn_(e) is absorbed in the bodymuch better than naturally-occurring zinc. In certain embodiments, thedisclosed composition contains zinc that is at least 80% ⁶⁴Zn_(e), atleast 90% ⁶⁴Zn_(e), at least 95% ⁶⁴Zn_(e), or at least 99% ⁶⁴Zn_(e), forexample, zinc that is 80% ⁶⁴Zn_(e), 85% ⁶⁴Zn_(e), 90% ₆₄Zn_(e), 95%⁶⁴Zn_(e), 99% ⁶⁴Zn_(e), or 99.9% ⁶⁴Zn_(e).

In another aspect, this disclosure provides a method of treating and/orpreventing obesity and/or overweightness by administering atherapeutically or prophylactically effective amount of a disclosedcomposition to a subject in need thereof.

The disclosed compositions may be administered to a subject to decreasethe subject's weight or to prevent or decrease weight gain. In someembodiments, the subject may be overweight or obese. In someembodiments, the subject may be someone who wishes to avoid becomingoverweight or obese. The subject may be a human or a non-human mammal,such as a non-human primate or a domesticated dog or cat.

A method is provided of preventing or treating overweightness or obesitycomprising administering to a subject in need thereof a prophylacticallyor therapeutically effective amount of a composition comprising a⁶⁴Zn_(e) compound or a salt thereof. A method is provided to decreasethe levels of triglycerides, cholesterol and free fatty acids in theserum of a subject comprising administering to a subject in need thereofan effective amount of a composition comprising a therapeuticallyeffective amount of a ⁶⁴Zn_(e) compound or a salt thereof. A method isprovided to decrease the levels of pro-inflammatory cytokines in theserum and adipose tissue of a subject comprising administering to asubject in need thereof an effective amount of a composition comprisinga therapeutically effective amount of a ⁶⁴Zn_(e) compound or a saltthereof. In some embodiments, the composition further comprises adiluent or an excipient. In some embodiments, the diluent is water. Infurther embodiments, the water diluent is deuterium-depleted water. Insome embodiments, the ⁶⁴Zn_(e) compound or a salt thereof is between20-100% ⁶⁴Zn_(e). In further embodiments, the ⁶⁴Zn_(e) compound or asalt thereof is at least 80% ⁶⁴Zn_(e). In further embodiments, the⁶⁴Zn_(e) compound or a salt thereof is at least 95% ⁶⁴Zn_(e). In someembodiments, the composition contains between 0.05 mg and 110 mg of⁶⁴Zn_(e). In some embodiments, wherein the composition contains between1 and 10 mg of ⁶⁴Zn_(e). In some embodiments, the ⁶⁴Zn_(e) compound or asalt thereof is at least 90% ⁶⁴Zn_(e) and the composition is an aqueoussolution in which ⁶⁴Zn_(e) is present at a concentration of between 0.1mg/ml and 10 mg/ml. In some embodiments, the ⁶⁴Zn_(e) is in a form ofsalt selected from the group consisting of aspartate (chemicalformula—(C₄H₅O₄N)₂ ⁶⁴Zn_(e)) with 2 aspartic acid molecules, sulfate,and citrate. In some embodiments, the composition is administered byinjection. In other embodiments, the composition is administered orally.In certain embodiments, the proinflammatory cytokines is one or more ofIL-1, IL-6, IL-12, and IFN-γ.

Formulating and Administering Compositions

The disclosed composition may be administered to a subject in needthereof by any suitable mode of administration, any suitable frequency,and at any suitable, effective dosage.

In some embodiments, the total amount of ⁶⁴Zn_(e) administered is thesame as the U.S. recommended daily allowance or intake of zinc. In someembodiments, the total amount of ⁶⁴Zn_(e) administered is ½, twice,three times, five times, or ten times the U.S. recommended dailyallowance or intake of zinc. In some embodiments, the total amount of⁶⁴Zn_(e) is between ½ and 10 times the U.S. recommended daily allowanceor intake of zinc. A disclosed composition may comprise the prescribeddaily amount to be administered once a day or some fraction thereof tobe administered a corresponding number of times per day. A disclosedcomposition may also comprise an amount of ⁶⁴Zn_(e) to be administeredonce every two days, once every three days, once a week, or at any othersuitable frequency.

The disclosed composition may be in any suitable form and may beformulated for any suitable means of delivery. In some embodiments, thedisclosed composition is provided in a form suitable for oraladministration, such as a tablet, pill, lozenge, capsule, liquidsuspension, liquid solution, or any other conventional oral dosage form.The oral dosage forms may provide immediate release, delayed release,sustained release, or enteric release, and, if appropriate, comprise oneor more coating. In some embodiments, the disclosed composition isprovided in a form suitable for injection, such as subcutaneous,intramuscular, intravenous, intraperitoneal, or any other route ofinjection. In some embodiments, compositions for injection are providedin sterile and/or non-pyrogenic form and may contain preservativesand/or other suitable excipients, such as sucrose, sodium phosphatedibasic heptahydrate or other suitable buffer, a pH-adjusting agent suchas hydrochloric acid or sodium hydroxide, and polysorbate 80 or othersuitable detergent.

When provided in solution form, in some embodiments, the disclosedcomposition is provided in a glass or plastic bottle, vial or ampoule,any of which may be suitable for either single or multiple use. Thebottle, vial or ampoule containing the disclosed composition may beprovided in kit form together with one or more needles of suitable gaugeand/or one or more syringes, all of which preferably are sterile. Thus,in certain embodiments, a kit is provided comprising a liquid solutionas described above, which is packaged in a suitable glass or plasticbottle, vial or ampoule and may further comprising one or more needlesand/or one or more syringes. The kit may further comprise instructionfor use.

In certain embodiments, the dosage of ⁶⁴Zn_(e) is proportional tovarious authoritative daily ingestion guidances (e.g. recommendeddietary allowance (USRDA), adequate intake (AI), recommended dietaryintake (RDI)) of the corresponding element. In some embodiments, thelight isotope dosage is between about ½ and about 20 times the guidanceamount, more preferably between about 1 and about 10 times the guidanceamount, even more preferably between about 1 and about 3 times theguidance amount. Thus, in certain embodiments, a single dose of adisclosed composition for daily administration would be formulated tocomprise a quantity within these ranges, such as about ½, about 1, about3, about 5, about 10, and about 20 times the guidance amount. Theseamounts generally are for oral intake or topical application. In someembodiments, the intravenous dosage is lower, such as from about 1/10 toabout ½ the guidance amount. Doses at the low end of these ranges areappropriate for anyone with a heightened sensitivity to a specificelement or class of elements (e.g., those with kidney problems). Forzinc, the daily guidance amount ranges from 2 mg in infants to 8-11 mg(depending on sex) for ages 9 and up. Daily dosages discussed throughoutthis application may be subdivided into fractional dosages and thefractional dosages administered the appropriate number of times per dayto provide the total daily dosage amount (e.g. ½ the daily doseadministered twice daily, ⅓ the daily dose administered three timesdaily, etc.). See Table 1.

TABLE 1 Element/Isotope guidance amount, daily Zinc/⁶⁴Zn_(e) Birth to 6months  2 mg 7 months-3 years  3 mg Children 4-8 years  5 mg Children9-13 years  8 mg 14-18 years (boys) 11 mg 14-18 years (girls)  9 mgAdults (men) 11 mg Adults (women)  8 mg

The disclosed composition can be produced by methods employed inaccordance with general practice in the pharmaceutical industry, suchas, for example, the methods illustrated in Remington: The Science andPractice of Pharmacy (Pharmaceutical Press; 21st revised ed. (2011)(hereinafter “Remington”).

In some embodiments, the disclosed compositions comprise at least onepharmaceutically acceptable vehicle or excipient. These include, forexample, diluents, carriers, excipients, fillers, disintegrants,solubilizing agents, dispersing agents, preservatives, wetting agents,preservatives, stabilizers, buffering agents (e.g. phosphate, citrate,acetate, tartrate), suspending agents, emulsifiers, and penetrationenhancing agents such as DMSO, as appropriate. The composition can alsocomprise suitable auxiliary substances, for example, solubilizingagents, dispersing agents, suspending agents and emulsifiers.

In certain embodiments, the composition further comprises suitablediluents, glidants, lubricants, acidulants, stabilizers, fillers,binders, plasticizers or release aids and other pharmaceuticallyacceptable excipients.

A complete description of pharmaceutically acceptable excipients can befound, for example, in Remington's Pharmaceutical Sciences (Mack Pub.,Co., N.J. 1991) or other standard pharmaceutical science texts, such asthe Handbook of Pharmaceutical Excipients (Shesky et al. eds., 8th ed.2017).

In some embodiments, the disclosed composition can be administeredintragastrically, orally, intravenously, intraperitoneally orintramuscularly, but other routes of administration are also possible.

Water may be used as a carrier and diluent in the composition. The useof other pharmaceutically acceptable solvents and diluents in additionto or instead of water is also acceptable. In certain embodiments,deuterium-depleted water is used as a diluent.

Large macromolecules that are slowly metabolized, such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, copolymers of amino acids, can also be used as carrier compoundsfor the composition. Pharmaceutically acceptable carriers in therapeuticcompositions may additionally contain liquids, such as water, saline,glycerol or ethanol. Moreover, the said compositions may furthercomprise excipients, such as wetting agents or emulsifiers, bufferingsubstances, and the like. Such excipients include, among others,diluents and carriers conventional in the art, and/or substances thatpromote penetration of the active compound into the cell, for example,DMSO, as well as preservatives and stabilizers.

The disclosed composition may be presented in various dosage formsdepending on the object of application; in particular, it may beformulated as a solution for injections.

The disclosed composition may be administered systemically. Suitableroutes of administration includes, for example, oral or parenteraladministration, such as intravenous, intraperitoneal, intragastric aswell as via drinking water. However, depending on a dosage form, thedisclosed composition may be administered by other routes.

In certain embodiments, the disclosed composition comprising ⁶⁴Zn_(e) isadministered intragastrically at a concentration of 2.25 mg/ml forpreventing and/or treating overweightness or obesity in an animalsubject. In further embodiments, the disclosed composition is about 2ml. In further embodiments, the level of enrichment of ⁶⁴Zn_(e) is about99% or more. In other further embodiments, the ⁶⁴Zn_(e) of the 2 mlcomposition comprises or consists of zinc aspartate (chemicalformula—(C₄H₅O₄N)₂ ⁶⁴Zn_(e)) with 2 aspartic acid molecules. The dose ofthe disclosed composition may vary depending on the subject beingtreated, severity of the disease, the patient's condition and otherfactors that will be taken into account by a person skilled in the artwhen determining the dosage and route of administration for a particularpatient based on his/her knowledge in the art.

Light isotopes may be purchased. Zn-64 oxide with the necessary degreeof enrichment may be purchased from, for example, Oak Ridge Nationallaboratory, Oak Ridge, Tenn., USA.

Zinc aspartate has a chemical formula—C₄H₅O₄N)₂ ⁶⁴Zn_(e), with 2aspartic acid molecules. The structure of aspartate is:

In certain embodiments, the disclosed composition comprises ⁶⁴Zn_(e) atabout 20% to to about 100% of the composition.

The disclosed composition comprising ⁶⁴Zn_(e) is metabolized in the bodymuch better than compositions comprising natural zinc in a form of saltsor chelates (which are not enriched for ⁶⁴Zn) that are conventionallyused in the art. In addition, the said composition helps reduce thetoxic effects inherent in traditional medicines with a comparable levelof efficacy in preventing and treating obesity.

The disclosed composition can be co-administered with another agent ortherapy.

A high efficacy of the claimed composition for the prevention andtreatment of obesity was demonstrated in an in vivo experiment involvingrat models.

EXAMPLES

For this invention to be better understood, the following examples areset forth. These examples are for purposes of illustration only and arenot be construed as limiting the scope of the invention in any manner.

Example 1 Comparison between ⁶⁴Zn_(e) and Naturally Occurring Zinc

A comparative study of the potential effects of ⁶⁴Zn_(e) and Znacetate—Zn(CH₃COO)₂ on the absorption and utilization of glucose by thebody showed that the zinc isotope had a better effect on the glucoseabsorption and utilization by the body on a number of parameters:

Positive dynamics of weight gain in control animals (vs the group ofanimals that were injected with Zn acetate (Zn(CH₃COO)₂) was recorded(FIG. 1).

On the seventh day after discontinuation of ⁶⁴Zn_(e) insulin levels inthe blood of animals increased (vs the group of animals that wereinjected with Zn acetate (Zn(CH₃COO)₂) (FIG. 3).

A significant increase was observed in the area of pancreatic islets inexperimental animals (microscopical examination on the 7^(th) day afterthe last administration of the substances) (vs the group of animals thatwere injected with Zn acetate (Zn(CH₃COO)₂) (FIG. 4). This is positivedynamics, as with the development of type I diabetes there is asignificant lack of insulin due to problems with its synthesis by theseislets. The obtained results correlate with the results on determininginsulin levels in the bloodstream (FIG. 3).

The glucose tolerance test showed a decrease in glucose levels in theanimals after administration of the zinc isotope compared with thecontrol group and the group of animals injected with Zn acetate(Zn(CH₃COO)₂) whose glucose levels also dropped, though not so strongly.This may indicate that the zinc isotope has an effect on the insulinlevels in blood, which in turn leads to the launch of mechanismsassociated with the utilization of glucose from the bloodstream.Considering that insulin is a zinc-dependent protein, it can be assumedthat the administration of zinc leads to an increase in either theactivity of this protein in relation to its receptor in tissues, or toan increase in the amount of this hormone in the bloodstream.

TABLE 2 Glucose tolerance test (fast overnight, glucose at the dose of 3g/kg body weight in a volume of 2 ml) 0 min 60 min Animal (1 h after (1h after weight, effector glucose g administration) administration)Control (2 ml saline) 1 190 4, 7 7, 9 2 196 5, 8 8  3 217 5, 1 8  4 2065, 4 8, 2 5 190 4, 8 7, 6 ⁶⁴Zn_(e) (dose: 5 mg/kg in a volume of 2 ml) 1195 4, 5 6  2 178 4, 8 6, 3 3 187 4, 5 6, 5 4 186 4, 7 6, 6 5 184 4, 86, 5 Zn acetate (dose: 5 mg/kg in a volume of 2 ml) 1 193 4, 6 7, 2 2186 5, 2 6, 9 3 192 5, 2 7, 3 4 188 4, 9 7, 2 5 195 5, 1 6, 8

Results obtained on type I diabetes model show that this substance has amore positive effect on the course of development of type I diabetes(vs. Zn acetate (Zn(CH₃COO)₂)) and can potentially be used to reducetoxic effects of increased glucose levels in the bloodstream during thedevelopment of this pathology.

Analysis of the accumulation of metals in the kidney and liver tissues(zinc, manganese and copper) showed that only zinc significantlyincreased in both groups of animals that were injected with zinc (Znacetate (Zn(CH₃COO)₂) or ⁶⁴Zn_(e)) both on day 1 and on day 7 afterdiscontinuation of the substances. This indicates that zinc injected toanimals accumulated and its utilization by the body did not increase.All other analyzed metals were within the same concentrations as in thecontrol group of animals. The data obtained indicate the absence of anynegative effects of the ⁶⁴Zn_(e) on the accumulation and utilization ofzinc and associated metals by the body.

All these data suggest a more pronounced and higher quality effect ofthe ⁶⁴Zn_(e) on absorption and utilization of glucose by the body incomparison with Zn acetate (Zn(CH₃COO)₂).

In this Example, Zinc acetate (natural zinc) was administered to anexperimental group of animals, at a dose of 3750 mcg of zinc (by metal)per 1 kg of body weight of the animal (rat). Zinc-64 in the form of zincaspartate was also administered to the experimental group of animals, ata dose of 3750 mcg of zinc (by metal) per 1 kg of body weight of theanimal (rat). The administration of these compositions was byintraperitoneal route.

Example 2 Anthropometric Effects of ⁶⁴Zn_(e)-Based Composition in AnimalModels of Obesity

To assess the effects of the ⁶⁴Zn_(e)-based composition on thedevelopment of obesity induced by high-fat diet, some anthropometricvalues in untreated animal models of obesity and animal models ofobesity treated with ⁶⁴Zn_(e) solution were evaluated.

White non-pedigree rats with an initial weight of 195-205±10 g were usedin the experiment. The animals were maintained in an accredited vivariumof the Academic and Research Center “Institute of Biology and Medicine”of Taras Shevchenko National University of Kyiv in accordance with theStandard rules on the arrangement, equipment and maintenance ofexperimental biological clinics (vivariums). The study was carried outin compliance with international standards and recommendations of theEuropean Convention for the Protection of Vertebrate Animals used forExperimental and other Scientific Purposes (Strasbourg, 18/03/1986) andapproved by the Bioethics Commission of the Academic and Research Center“Institute of Biology and Medicine”.

Statistical processing of the results was carried out using the methodsof variation statistics and correlation analysis using OrginLab Orgin®Pro 9.1 and StatSoft STaStica® 10 software (Brandt, Z. Statisticalmethods for analysis of observations. M.: Mir, 1975.-312p.). Thehypothesis of normal distribution of samples was tested using theShapiro-Wilk test. If a sample met the criteria of normal distribution,significance of differences between samples was determined using theStudent's t-test. If a sample did not meet the criteria of normaldistribution, significance of differences between samples was determinedusing the Mann-Whitney U test. Differences were considered statisticallysignificant when p<0.05.

Before the start of the experiment, animals were maintained on astandard diet of the vivarium. To induce obesity in experimentalanimals, they were fed high-fat diet which consisted of standard feed(60%), lard (10%), chicken eggs (10%), sucrose (9%), peanuts (5%), drymilk (5%) and sunflower oil (1%) (1%) (see X. H. Shen et al., Exp. Biol.and Med. 235: 47-51 (2010)). The feed was prepared by the presentinventors. The first 4 weeks of the experiment, all animals weremaintained on a high-fat diet after which they were randomly dividedinto two experimental groups:

animals in the first group (obesity) continued eating their high-fatdiet and had free access to water for the next 6 weeks of theexperiment;

animals in the second group (obesity+⁶⁴Zn_(e) solution) also followedtheir high-fat diet and had free access to water for the next 6 weeks ofthe experiment but every third day and until the end of the experimentthey were administered a solution of ⁶⁴Zn_(e) intragastrically at aconcentration of 2.25 mg/ml in a volume of 2 ml. This solution contained2.25 mg/ml of a pharmaceutically acceptable zinc salt, particularly zincaspartate, wherein the level of enrichment by ⁶⁴Zn was not less than 80percent. For the preparation of composition claimed standard Dulbecco'sphosphate-buffered saline from (specified a manufacturer) (based ondeuterium-depleted water Langway) as a diluent (liquid vehicle) wasused.

There was also a group of animals (control) that received standard dietprepared by the vivarium and had free access to water during the entireexperiment.

Animals in all groups were weighed once a week after an overnight fast.The amount of feed to be consumed by the animals was determined daily.At the end of a 10-week period of the development of obesity models, 24hours after the last administration of the zinc isotope solution,animals were removed from their cages and decapitated.

The body mass index (BMI) (the ratio of body weight (g) to the square ofbody length (cm²)) was calculated at the end of the experiment. Anincrease in BMI is a characteristic morphological sign of obesitydeveloping as a result of accumulation and redistribution of adiposetissue in the body. BMI makes it possible to assess the ratio of bodyweight to height (body length) and thereby indirectly assess whether theweight is insufficient, normal or excessive. In addition, BMI is used asan integral value that characterizes a composition of the body and adegree of fat deposits, because the distribution of adipose tissue inthe body determines the risk of metabolic complications associated withobesity, which must be considered when examining patients that developobesity. BMI is not only a diagnostic criterion for obesity, but also agood measure of a patient's risk for diseases that can occur withoverweight and obesity.

The data obtained during the experiment (Table 3) show that on the10^(th) week of the experiment, the mean body mass index of controlanimals was 0.60 g/cm² which value is within a reference range foranimals of this age group. BMI of animals eating high-fat diet was 1.14times higher than BMI of animals of the control group (0.71 g/cm²). Thebody mass index of rats receiving the ⁶⁴Zn_(e) solution during theexperiment was lower than that of untreated animal models of obesity butslightly higher than the control value (0.65 g/cm²), which indicatesthat the ⁶⁴Zn_(e) solution has a positive effect on the generalmetabolic status of animals.

TABLE 3 Some anthropometric values, the amount and calorie content offood (M ± m, n = 10) Experimental groups C + DIO + C zinc DIO zinc BMI(g/cm²) 0.60 0.59 0.71 0.65 Weight gain as of the 59 59 103 62 end ofthe experiment (%) Amount of food 34 32 35 29 consumed (g/day) Caloriecontent 525 490 1001 823 of food (kJ/day) Note: C: control; C + zinc:control on the background of ⁶⁴Zn_(e) administration; DIO: diet-inducedobesity; DIO + zinc: diet-induced obesity on the background of ⁶⁴Zn_(e)administration.

Since BMI is calculated based on weight, a decrease in BMI value may bedirectly related to the lower weight of animals that received the⁶⁴Zn_(e) solution. Therefore, it was further investigated whetheradministration of the ⁶⁴Zn_(e) solution had an effect on the weight andweight gain of animal models of obesity. The data obtained (FIG. 1) showthat the dynamics of weight gain by animals of the experimental groupsdiffered significantly. Thus, animals that were maintained on a high-fatdiet and received the ⁶⁴Zn_(e) solution gained less weight than animalsthat were only fed a high-fat diet. Particularly noticeable differencein the weight gain of animals of both groups was observed starting fromthe 4th week of the experiment. An increase in the body weight ofanimals eating a high-fat diet reached 103% by the end of theexperiment, while animals that received intragastric injections of the⁶⁴Zn_(e) solution gained not much more weight than animals in thecontrol group (62% vs. 59%).

It is known that the development of obesity, due to disruption of thecoordinated work of a number of neurotransmitter and hormonal systems inthe body, leads to disturbances at the level of appetite control andregulation of a feeling of satiety. These disturbances promote excessivefood intake and are often accompanied by the development of hyperphagia,a state of an abnormally great desire for food energy the equivalent ofwhich exceeds the energy needs of the body (L. Zhou et al., CellMetabolism 6: 398 (2007)).

To define possible mechanisms of decrease in the body weight gain ofanimals treated with the ⁶⁴Zn_(e) solution, the amount of food consumedby the animals was analyzed. The data obtained are presented in Table 3.

When the data calculated for all experimental groups were compared,there are no particular differences in the amount of food the animalsate on average per day. Thus, animals of the control group and the groupof obesity models consumed about 35 g of food per day. However, itshould be noted that animals in the control group were maintained on astandard diet while animals in the group of diet-induced obesity modelsconsumed specially prepared high-fat diet the calorie content of whichwas significantly higher.

Analysis of the results obtained, with due consideration of the caloriecontent of the food consumed by animals, shows a significant differencein values. Despite the same amount of food eaten by animals, the caloriecontent of food consumed by the group of diet-induced obesity modelsthat were administrated the composition claimed, was lower than thecalorie content for the control group of animals with diet-inducedobesity (without administration of the disclosed composition).Furthermore, on week 10, the caloric content for the group ofdiet-induced obesity models that were administrated the disclosedcomposition, was almost the same as for the control group which consumedstandard food, and for the group, which consumed standard food withsimultaneous administration of the disclosed composition.

The dynamics of calorie content of food consumed by animals during 10weeks of the experiment is shown in FIG. 2.

The data obtained suggest that the ⁶⁴Zn_(e)-based composition has aneffect on the feeling of satiety because, having free access to food,animals treated with the disclosed composition of a disclosed methodconsumed significantly less food compared with untreated animals thatwere only maintained on a high-fat diet. In other words, the animalsthat received the ⁶⁴Zn_(e) solution ate less and gained less weight thanthe animals that did not receive the ⁶⁴Zn_(e) solution. This differencecan be explained by both direct and indirect effects of zinc on energyhomeostasis.

Thus it was demonstrated that administration of the ⁶⁴Zn_(e)-basedcomposition caused a decrease in the amount of food consumed per day,which, accordingly, was accompanied by a less pronounced weight gain inanimals and normalization of their body mass index in comparison withsimilar values in untreated animal models of obesity.

Example 3 Biochemical Effects of ⁶⁴Zn_(e)-Based Composition in AnimalModels of Obesity

An experiment to study the effects of light zinc isotope ⁶⁴Zn_(e) onblood biochemical variables which undergo pathological changes inobesity, particularly the lipid profile, was carried out. To this end,high-fat diet obesity was induced in experimental animals as describedin Example 2. For the experiment, the following animals were used:control animals that consumed standard diet; animals that receivedhigh-fat diet for the next 6 weeks; and animals that were fed high-fatdiet but were also administered the ⁶⁴Zn_(e)-based composition (zincaspartate with ⁶⁴Zn_(e) with an enrichment of 80% or more at aconcentration of 2.25 mg/ml was administered intragastrally in a volumeof 2 ml) during all 6 weeks of the experiment. The results of theexperiment are shown in Table 4.

TABLE 4 Serum biochemical variables in animals from experimental groups(M ± m, n = 10) Groups Variables C DIO DIO + ⁶⁴Zn_(e) Alkalinephosphatase 74.3 ± 12.1  37.2 ± 15.4*  87.6 ± 18.7# activity, RUTriglycerides, g/L 2.55 ± 0.20  4.39 ± 0.73*  2.79 ± 0.30# Cholesterol,mmol/L 2.42 ± 0.19  5.76 ± 0.87*  2.83 ± 0.23# Free fatty acids, mg/L23.60 ± 4.67  74.50 ± 9.23* 31.62 ± 7.92# *the difference is significantcompared to the control group of animals; #the difference is significantcompared to the group of animal models of obesity Note: C: control; DIO:diet induced obesity; DIO + ⁶⁴Zn_(e): diet induced obesity on thebackground of ⁶⁴Zn_(e) administration.

It was found that the ⁶⁴Zn_(e)-based composition had a positive effecton lipid metabolism in the body. A decrease in the levels oftriglycerides, cholesterol and free fatty acids in the serum of animalsthat were fed a high-fat diet and treated with ⁶⁴Zn_(e) was almost atthe same level as in the control group of animals.

Example 4 Effects of ⁶⁴Zn_(e) on the Redox State in Experimental Animals

A number of studies have shown that obesity is closely associated withan altered redox state and an increased metabolic risk. It is oxidativestress that is one of the factors causing adipocyte dysfunction.Oxidative stress and the resulting tissue damage and cell death are thebasis for the development of many chronic pathological conditions.Excessive production of free radicals and/or depletion of theirdetoxification system lead to the prooxidant-antioxidant imbalance,which in turn affects the structures of cellular membrane lipids andproteins and nucleic acids. Lipid peroxidation (LPO) mediated by freeradicals is one of the important causes of the destruction of cellularmembranes and further cell damage. Degradation of membrane lipidsinduces an increase in the membrane's fluidity and its permeability toions, which disrupts cellular homeostasis as a whole. Products of freeradical oxidation (4-hydroxyalkenes, malonic dialdehyde, etc.) arehighly mutagenic and cytotoxic.

In addition, oxidative stress activates preadipocyte differentiation andstimulates hypertrophy of mature adipose cells. Excessive production ofROS in the accumulated adipose tissue further leads to the induction ofoxidative stress in the bloodstream, which contributes to the spread ofoxidative stress to organs distant from the fat depot.

The prooxidant-antioxidant balance in animals was assessed using theobesity models as described in Example 2. The control group, the groupof untreated animal models of obesity and the group of animals that werefed a high-fat diet and treated with ⁶⁴Zn_(e) were used in theexperiment.

Concentrations of lipid peroxidation products serve as an informativecriterion making it possible to draw a conclusion about intensity ofoxidative processes. There are primary lipid peroxidation products (suchas conjugated dienes) and secondary lipid peroxidation products (such asaldehydes, malonic aldehyde in particular), which are formed as a resultof breakdown of carbon-carbon double bonds in the carbon skeletons ofoxidized molecules. Subsequently, the LPO initiation leads to theformation of conjugated Schiff bases of phospholipids andmalonaldehyde-like products, which cause disturbances in the orderedorientation of phospholipid molecules and affect lipoproteinintermolecular interactions and configuration of the basement membrane.

Considering the above, concentrations of primary LPO products(conjugated dienes (CD)), secondary LPO products (TBA-reactivesubstances (TBARSs)) and end LPO products (Schiff bases (SB)) in animalstreated with ⁶⁴Zn_(e) were determined. Taking into account that obesityis accompanied by the development of systemic oxidative stress whichcovers most tissues to various extents and leads to the disruption ofintegrity of cellular membranes and admission of lipid peroxidationproducts to the bloodstream, values characterizing the state of theprooxidant-antioxidant system were determined in the blood serum ofanimals.

It was found that the obesity models had elevated serum levels ofprimary products of free radical lipid oxidation (1.86 times as high asin the control) (Table 5). Such result can be explained from thestandpoint of disturbed lipid metabolism, impairment of the processes oftransportation of fatty acids in particular, and, accordingly, anincrease in the plasma levels of free and esterified fatty acids, whichare direct substrates for the action of reactive oxygen species.

On the other hand, accumulation of lipid peroxidation products in serummay be a direct result of violation of the integrity of cellularmembranes due to oxidative destruction of their lipid component andadmission of lipid peroxidation products to the bloodstream.

TABLE 5 Serum levels of lipid peroxidation products in animals fromexperimental groups (M ± m, n = 10) TBA-reactive substances, Conjugatednmol/mg protein dienes, Fe²⁺-ascorbate- Experimental nmol/mg Spontaneousinduced Schiff bases, groups protein accumulation accumulation RU/mgprotein C  0.021 ± 00.001 0.006 ± 0.0003 0.033 ± 0.005 41.31 ± 2.47 DIO  0.039 ± 0.002 * 0.029 ± 0.002*   0.61 ± 0.003* 168.86 ± 8.15* DIO +⁶⁴Zn_(e) 0.025 ± 0.008  0.005 ± 0.0003#     0.15 ± 0.008*, #    56.27 ±4.33*, # *the difference is significant compared to the control group ofanimals; #the difference is significant compared to the group of animalmodels of obesity Note: C: control; DIO: diet induced obesity; DIO +⁶⁴Zn_(e): diet induced obesity on the background of ⁶⁴Zn_(e)administration.

Thus, elevated levels of lipid peroxidation products on the 10^(th) weekof obesity development clearly indicate that oxidative stress has asystemic nature and that this process is chronic, which is anunfavorable prognostic marker as these metabolites are extremely toxiccompounds and their negative impact is exhibited at different levels andleads to DNA molecule damage, destruction of protein molecules andglycosaminoglycans, changes in the lipid composition of cellularmembranes and disruption of membrane-associated processes.

Administration of ⁶⁴Zn_(e)-based composition to animals helped normalizethe levels of primary, secondary and end LPO products, which serves asadditional evidence of the ability of ⁶⁴Zn_(e) to influence an overallprooxidant-antioxidant status of the body.

According to modern concepts, reactive oxygen species not only activatelipid peroxidation processes but also cause oxidative destruction ofprotein molecules, causing disruption of conformation of both solubleand membrane-bound enzymes, receptors and ion channels, which ultimatelyleads to the loss of their biological activity (enzymatic, receptor,transport, for example). Protein oxidation results in the formation ofaldehyde and ketone groups of amino acid residues (carbonyl groups) inproteins.

Thus, an increase in the number of oxidatively modified proteins may beconsidered as an early criterion of free radical tissue damage and amarker of the depletion of antioxidant defense system in the body. Thisstudy revealed increased serum levels of oxidatively modified proteinsin animal models of obesity (Table 6).

TABLE 6 Serum levels of products of oxidative modification of proteinsin animals from experimental groups (M ± m, n = 10) Aldehyde- Ketone-dinitrophenyl- dinitrophenyl- Groups hydrazones, hydrazones, nmol/mgprotein nmol/mg protein C 0.187 ± 0.009 0.255 ± 0.023 DIO  0.698 ±0.041*  0.571 ± 0.035* DIO + ⁶⁴Zn_(e)    0.253 ± 0.012*, #    0.200 ±0.024*, # *the difference is significant compared to the control groupof animals; #the difference is significant compared to the group ofanimal models of obesity Note: C: control; DIO: diet induced obesity;DIO + ⁶⁴Zn_(e): diet induced obesity on the background of ⁶⁴Zn_(e)administration.

The experimental data showed that in animals that were fed a high-fatdiet during the entire experiment and received injections of the⁶⁴Zn_(e) solutions, the levels of aldehyde-dinitrophenyl-hydrazonesexceeded the benchmark but were lower compared to the values inuntreated animals having obesity. As forketone-dinitrophenyl-hydrazones, their concentration remained within thecontrol value. Such results correlate with the data showing a decreasein the levels of LPO products and suggest a decrease in the intensity offree radical oxidation reactions.

Example 5 Effects of ⁶⁴Zn_(e)-Based Composition on Cytokine Profile inAnimal Models of Obesity

The cytokine profile in animals was assessed using the obesity models asdescribed in Example 2. The control group, the group of untreated animalmodels of obesity and the group of animals that were fed a high-fat dietand treated with ⁶⁴Zn_(e) were used in the experiment.

Obesity pathogenesis is accompanied by a systemic chronic inflammatoryprocess, the degree of intensity of which can be assessed by the serumlevels of pro- and anti-inflammatory cytokines.

Analysis of the serum cytokine profile in animal models of obesityshowed an increase in the levels of pro-inflammatory cytokines (Table7). In animals fed a high-fat diet and administered the ⁶⁴Zn_(e)solution, there was a decrease in the serum levels of pro-inflammatorycytokines against the background of an increase in the levels ofanti-inflammatory cytokines, which were even higher than in the animalsfrom the control group.

TABLE 7 Serum cytokine profile in animals from experimental groups (M ±m, n = 10) Levels, RU/mg protein Pro-inflammatory cytokines Groups IL-1IL-6 IL-12 IFN-γ C 3.4 ± 0.3 4.5 ± 0.3 0.5 ± 0.05 3.6 ± 0.8 C + ⁶⁴Zn_(e)3.5 ± 0.7 4.3 ± 0.2 0.3 ± 0.04 4.6 ± 0.6 DIO 11.1 ± 2.0*  7.9 ± 0.5* 3.7 ± 0.07*  6.5 ± 0.8* DIO + ⁶⁴Zn_(e)  4.2 ± 0.4#  5.1 ± 0.4#    2.4 ±0.06*, # 4.1 ± 1.2 Levels, RU/mg protein Anti-inflammatory cytokinesGroups IL-4 IL-10 TGF C 5.1 ± 0.2 3.9 ± 0.4 3.8 ± 0.8 C + ⁶⁴Zn_(e) 4.6 ±0.8 4.1 ± 0.5 4.1 ± 0.4 DIO 4.4 ± 0.9 4.1 ± 1.5 3.5 ± 1.3 DIO + ⁶⁴Zn_(e)5.6 ± 1.6   6.8 ± 1.1*, #   5.7 ± 0.3*, # *the difference is significantcompared to the control group of animals; #the difference is significantcompared to the group of animal models of obesity Note: C: control; DIO:diet induced obesity; DIO + ⁶⁴Zn_(e): diet induced obesity on thebackground of ⁶⁴Zn_(e) administration.

One of the basic mechanisms of the effects of zinc enriched for theisotope ⁶⁴Zn_(e) on the cytokine profile may be its inhibition oftranscription factors sensitive to oxidative stress. A certainnormalizing effect of the ⁶⁴Zn_(e)-based composition on the cytokineprofile in animal models of obesity may serve as evidence of a possibleanti-inflammatory potential of the claimed composition in obesity.

Thus, the experimental data confirmed positive effects of the⁶⁴Zn_(e)-based composition on a number of pathological variables inanimal models of obesity. In particular, it was demonstrated thatadministration of the ⁶⁴Zn_(e)-based composition to experimental animalscaused a decrease in the body mass index and a reduction in the bodyweight gain and the amount of food consumed; ⁶⁴Zn_(e) was found to havea positive effect on lipid metabolism in the bodies of animals;normalization of prooxidant-antioxidant homeostasis due to a decrease inthe intensity of free radical processes was demonstrated; the ability of⁶⁴Zn_(e) to influence the serum cytokine profile in animals wasrevealed. The effects observed in this study support the efficacy of theclaimed ⁶⁴Zn_(e)-based composition for the prevention and treatment ofobesity.

For Example 2-5, the zinc-64 enriched disclosed composition has a zincsalt/compound with the following structural formula:

The compound is a crystalline hydrate, which contains 2 water molecules.The molar mass is 364 g/mol. 2,2 H₂O should be considered as .2 H₂O,Because the 0.2 H₂O is unbound water that can evaporate when drying thepowder. .2 H₂O is crystalline hydrate and is a part of the molecule. 4.5mg of zinc aspartate (which was in a solution volume of 2 ml) was used,which contained 17.8% pure zinc-64 (by metal). Thus, each dose, whichwas 4.5 mg zinc aspartate, contained 800 ug zinc-64 (by metal).

Example 6 Zn64 Stable Isotope in Aspartate Form on Obesity and Type 2Pre-Diabetes in Experimental Animals (Rats) Fed High-Fat Diets Over aSpecified Time

List of Abbreviations

ROS—reactive oxygen species

AOD—antioxidant defense

FFA—free fatty acids

GI tract—gastrointestinal tract

BMI—body mass index

IDO—indoleamine-2,3-dioxygenase

IR—insulin resistance

MAO—monoamine oxidase

OMP—oxidative modification of proteins

OS—oxidative stress

SOD—superoxide dismutase

IL—interleukin

This study assesses the effects of Zn-64 stable isotope in aspartateform on the development of obesity induced by high-fat diet inexperimental animals. The following tasks were set:

To investigate the effects of Zn-64 stable isotope in aspartate form ona number of anthropometric (body mass index, weight, weight gain) andbiochemical (glucose concentration, insulin level, alkaline phosphataseactivity, albumin content) values in animal models of obesity.

To investigate the effects of Zn-64 stable isotope in aspartate form onmorphofunctional properties of the pancreas and liver of animals fed ahigh-fat diet.

To assess the effects of Zn-64 stable isotope in aspartate form on thefunctions of central and peripheral serotoninergic systems (tryptophanand serotonin levels, tryptophan hydroxylase, tryptophan decarboxylase,monoamine oxidase and indoleamine 2,3-dioxygenase activity) in animalmodels of obesity.

To assess the effects of Zn-64 stable isotope in aspartate form on freeradical processes (levels of primary, secondary and end products oflipid peroxidation, levels of products of oxidative modification ofproteins) and the activity of key antioxidant enzymes (superoxidedismutase, catalase) in serum and adipose tissue in animal models ofobesity.

To assess the effects of Zn-64 stable isotope in aspartate form on thecytokine profile (levels of pro- and anti-inflammatory cytokines) inserum and adipose tissue, as well as resistin and ghrelin levels inanimal models of obesity.

To investigate the effects of Zn-64 stable isotope in aspartate form onthe distribution of divalent metals (zinc, copper, manganese, etc.)between different organs in animal models of obesity.

Materials and Methods

Development of obesity models

White non-pedigree rats were used in the studies. The animals weremaintained in an accredited vivarium of the Academic and Research CenterInstitute of Biology and Medicine of Taras Shevchenko NationalUniversity of Kyiv in accordance with the Standard rules on thearrangement, equipment and maintenance of experimental biologicalclinics (vivariums). The study was carried out in compliance withinternational standards and recommendations of the European Conventionfor the Protection of Vertebrate Animals used for Experimental and otherScientific Purposes (Strasbourg, Mar. 18, 1986) and approved by theBioethics Commission of the Academic and Research Center Institute ofBiology and Medicine. Murzin, O. B., European Convention for theProtection of Vertebrate Animals Used for Experimental and OtherScientific Purposes/O. B. Murzin, //Practical workbook on humanphysiology.—Dnipropetrovsk: Publishing House of DnipropetrovskUniversity, 2004.-P. 135-148.

Animals with an initial weight of 195-205±10 g maintained on thestandard diet of the vivarium before performing obesity models were usedin the experiments. To induce obesity in experimental animals, they werefed high-fat diet which consisted of standard feed (60%), lard (10%),chicken eggs (10%), sucrose (9%), peanuts (5%), dry milk (5%) andsunflower oil (1%), Shen X. et al., Experimental Biology andMedicine.-2010.-No 235.-P. 47-51. The first 4 weeks of the experiment,all animals were maintained on a high-fat diet, after which they wererandomly divided into two experimental groups:

-   -   animals in the first group (obesity) continued to eat their        high-fat diet and had free access to water for the next 6 weeks        of the experiment.    -   animals in the second group (obesity+solution of Zn-64 stable        isotope in aspartate form) also followed their high-fat diet and        had free access to water for the next 6 weeks of the experiment.        But every third day and until the end of the experiment they        were intragastrically administered a solution of Zn-64 stable        isotope in aspartate form. The dose of zinc aspartate        administered to each animal was 4.5 mg (substance per animal),        which were administered with a gavage in a volume of 2 ml of        solution.

There was also a group of animals (control) that received standard dietprepared by the vivarium and had free access to water during the entireexperiment.

To check the presence or absence of the effect of Zn-64 stable isotopein aspartate form on the studied anthropometric and biochemicalparameters, a group of animals was formed (control+solution of Zn-64stable isotope in aspartate form) which ate standard vivarium diet andhad free access to water over the entire period of the experiment butevery third day and until the end of the experiment the animals wereintragastrically administered Zn-64 stable isotope in aspartate form.The dose of zinc aspartate administered to each animal was 4.5 mg(substance per animal), which were administered with a gavage in avolume of 2 ml of solution.

Animals in all groups were weighed once a week after an overnight fast.The amount of feed to be consumed by the animals was determined daily.At the end of a 10-week period of the development of obesity models, 24hours after the last administration of Zn-64 stable isotope in aspartateform, animals were removed from their cages and decapitated.

The body mass index (BMI) (the ratio of body weight (g) to the square ofbody length (cm2)) was calculated at the end of the experiment.

Preparation of Blood Serum

Animal serum was prepared from whole blood. To remove fibrinogen-relatedproteins, the blood was incubated at 37° C. for 30 minutes, after whicha blood clot was carefully separated from the walls of the tube with aclean, dry glass rod to accelerate the production of serum. Samples werecentrifuged for 15 min at 2500 g. The resulting supernatant (serum) wasimmediately separated from blood cells and frozen and stored at −20° C.until the experiments.

Preparation of Adipose Tissue Homogenate

At the end of the experiment, the animals were euthanized bydecapitation. All manipulations during the tissue removal were carriedout at a temperature of 1-4° C.

Adipose tissue was crushed using cold scissors then transferred to ahomogenizer with a loose-fitting Teflon pestle. The tissue washomogenized using approximately 30 strokes of the pestle in coldhomogenization buffer (50 mM Tris-HCl (pH 7.4) containing 130 mM NaCl).Primary homogenate thus obtained was centrifuged at 600 g for 15minutes. The supernatant was carefully collected and re-centrifuged at15,000 g for 15 minutes. The supernatant was then frozen and stored at−80° C. until the experiments.

Preparation of Brain Tissue Homogenate

At the end of the experiment, the animals were euthanized bydecapitation. All manipulations during the organ removal were carriedout at a temperature of 1-4° C.

The animal's head was separated from the body and the skull wascarefully cut. The brain was carefully lifted with a scalpel from thebony vault, all cranial nerves were amputated and the brain was removedfrom the skull. The brain was divided into two parts with a longitudinalincision made between the hemispheres.

Brain tissue was crushed using cold scissors then transferred to ahomogenizer with a loose-fitting Teflon pestle. The tissue washomogenized using approximately 30 strokes of the pestle in coldhomogenization buffer (50 mM Tris-acetate, pH 7.4, containing 5 mM EDTAand 10% sucrose). The tissue:buffer ratio was 1:10. The homogenate thusobtained was centrifuged at 1500 g for 20 minutes. The supernatant wasthen carefully collected and frozen and stored at −80° C. until theexperiments.

Preparation of Duodenal Tissue Homogenate

At the end of the experiment, the animals were euthanized bydecapitation. All manipulations during the organ removal were carriedout at a temperature of 1-4° C.

After opening the abdomen, the duodenum was removed from the body of theanimal and washed in a Petri dish in 0.9% sodium chloride solution. Theduodenal mucosa was isolated mechanically using a scalpel and thenhomogenized in 10 mM Tris-HCl buffer, pH 7.4, containing 1 mM EDTA and0.25 M sucrose. The tissue : buffer ratio was 1:10. The homogenate thusobtained was centrifuged at 1500 g for 10 minutes. The supernatant wasthen carefully collected and frozen and stored at −80° C. until theexperiments.

Determination of Glucose Concentration in Serum

The glucose concentration was measured in the blood of animals that hadfasted for at least 2 hours. Blood was collected from the tail veinusing a catheter. The glucose concentration was determined using aGLUTOFOT-II glucose meter (Ukraine) according to the manufacturer'sinstructions. Medical test “Glyukofot-II”: [user's manual for“Glyukofot-II-Hemoglan].-Kiev: Norma, 2008.-12 p. The test stripcontained all the necessary reagent components to determine the glucoseconcentration by glucose oxidase method, including absorption of glucoseoxidase and peroxidase enzymes into a porous hydrophilic membrane.Formation of a colored complex was the result of the reaction. A drop ofwhole blood was applied to the strip and left at room temperature for 30seconds. The strip was then washed with distilled water and placed in ablood glucose meter. The glucose concentration was expressed in mmol/L.

Determination of the Levels of Insulin, Interleukins and Adipokines inSerum and Adipose Tissue Homogenate

The levels of insulin, interleukins and adipokines were determined usingimmunoenzyme method (Halenova T I et al. RSC Adv. 2016; 6: 100046-55),which was carried out in microplates with a sorption capacity inaccordance with the soluble protein test procedure. The antigensolution, previously diluted with 0.1 M NaHCO₃ buffer, pH 9.6, to aconcentration of 10 μg/ml, was incubated in the plate wells for 12 hoursat 4° C. Unbound material was removed by washing the wells three timeswith TBS buffer, first with 0.05% Tween-20, then without Tween-20.Non-specific binding sites were blocked by adding a solution of 5% skimmilk or a solution of 1% bovine serum albumin to the plate wells andincubating them for 60 minutes at 37° C. After incubation, the wellswere washed three times with TBS, first with 0.05% Tween-20, thenwithout Tween-20. Primary antibodies were diluted in TBS in accordancewith the manufacturer's instructions and incubated with the antigen for60 min at 37° C. After washing with TBS working buffer, first with theaddition of 0.05% Tween-20, then without Tween-20, the conjugate ofsecondary antibodies was added to the wells and incubated for 60 min at37° C. As with the primary antibodies, secondary antibodies were dilutedin TBS in accordance with the manufacturer's instructions. After thewashing procedure, phenyldiamine dihydrochloride as substrate in 0.05Mphosphate-citrate buffer was added to each well, followed by theaddition of 0.3% hydrogen peroxide. After 10 minutes, the reaction timeneeded for development, 2.5 n H₂SO₄ was added.

The absorbance at a wavelength of 492 nm was measured using a μQuantmicroplate spectrophotometer (BioTek Instruments).

Determination of Serum Alkaline Phosphatase Activity

Serum alkaline phosphatase activity in the animals was measuredspectrophotometrically using a Microlab 300 biochemical analyzer andstandard PLIVA-Lachema Diagnostika test kits (Czech Republic). Testkit//Pliva-Lachema Diagnostika.-2008.

As a result of hydrolytic cleavage of p-nitrophenyl phosphate catalyzedby alkaline phosphatase, p-nitrophenol is formed, which gives an intenseyellow color in alkaline medium. The optical density of samples wasmeasured at a wavelength of 405 nm. The alkaline phosphatase activitywas expressed in relative units.

Determination of Serum Albumin

The levels of serum albumin in the animals were determinedspectrophotometrically using a Microlab 300 biochemistry analyzer andstandard PLIVA-Lachema Diagnostika test kits (Czech Republic). Testkit//Pliva-Lachema Diagnostika.-2008.

Determination of Serum Superoxide Dismutase Activity

To measure superoxide dismutase activity, a method based on the abilityof this enzyme to inhibit auto-oxidation of adrenaline was used. SyrotaT. V. Questions of med. clin.-1999.-V. 5, No. 3.-P. 263-272.

Serum aliquots were added to microplate wells containing 0.2Mbicarbonate buffer, pH 10. The reaction was initiated by adding 0.1%adrenaline solution in each well. A relevant volume of buffer was addedto the “blank” wells to which no test sample was added. The opticaldensity was measured at a wavelength of 347 nm using a μQuant microplatespectrophotometer (BioTek Instruments) at the 4^(th) and 8^(th) minuteafter adrenaline was added to the wells. The enzyme activity wasexpressed in relative units/min/mg.

Determination of Catalase Activity

To determine catalase activity, a spectrophotometric method was usedwhich depends on the ability of hydrogen peroxide to form a stablecolored complex with molybdenum salts. Korolyuk M. A. et al., Lab.Business.-1988.-No. 1.-P. 44-67. During incubation, the concentration ofhydrogen peroxide decreased due to the enzymatic activity mediated bycatalase in the test sample. 4% ammonium molybdate solution and 0.03%hydrogen peroxide were used. The reaction was started by adding the testsample to 0.03% hydrogen peroxide. Instead of protein, an appropriatevolume of distilled water was added to the blank sample. The reactionwas stopped after 10 minutes by addition of 4% ammonium molybdatesolution to the incubation medium. The optical density was measured at awavelength of 410 nm using a μQuant microplate spectrophotometer (BioTekInstruments). Catalase activity was calculated using a calibration curveand quoted as μmol H₂O₂/mg protein×min.

Determination of the Levels of Diene Conjugates and Schiff Bases inSerum and Adipose Tissue Homogenates

An aliquot of the test sample containing 0.1-0.5 mg of protein wasplaced in a tight-fitting glass homogenizer, to which heptane/isopropylalcohol mixture was added at a 1:1 ratio, and homogenized for 10minutes. The samples then were centrifuged at 1000 g for 15 minutes intest tubes closed with a tight-fitting stopper. The supernatant fractionwas carefully collected and distilled water was added to separate thephases of heptane and isopropyl alcohol. Levels of Schiff bases weredetermined in the upper heptane phase by measuring the optical densityof samples at an excitation wavelength of 360 nm and an emissionwavelength of 420 nm using a spectrophotometer. The levels of Schiffbases represented the number of units per 1 mg of protein.

To determine the levels of diene conjugates, an aliquot of the heptanephase was taken to which 96% ethanol was added and the samples werethoroughly mixed. The optical density of the samples was measured at awavelength of 233 nm using a spectrophotometer. The levels of dieneconjugates were calculated using a molar extinction coefficient (2.2×10⁵cm⁻¹×M⁻¹) for conjugated dienes occurring when polyunsaturated higherfatty acids were oxidized and quoted in nmol per mg protein. NedzvetskyV et al., J Diabetes Metab. 2012; 3(8): 1-9.

Determination of Serum Levels of TBA-Active Products and Adipose TissueHomogenates

An aliquot of the test sample was added to the sample and an equalvolume of 17% trichloroacetic acid (TCA) was added. The samples werecentrifuged at 1000 g for 15 min. Nedzvetsky V et al., J Diabetes Metab.2012; 3(8): 1-9. A solution of 0.8% thiobarbituric acid was added to thesupernatant and incubated in a boiling water bath for 10 minutes beforethe color developed. The optical density of the samples was measured ata wavelength of 532 nm using a spectrophotometer. The concentration ofTBA-active products was expressed in nmol per 1 mg of protein and wascalculated using a molar extinction coefficient (1.56×10⁵ cm⁻¹×M⁻¹).

Determination of the Levels of Products of Oxidative Modification ofProteins

Estimation of the intensity of oxidative modification of proteins isbased on the reaction between protein carbonyls and Schiff bases and2,4-dinitrophenylhydrazine (DNPH) with the formation of2,4-dinitrophenylhydrazons of a neutral and basic nature. Vartanyan L.S, Gurevich S. M. Biochemistry.-1989.-Vol. 54, No. 6.-P. 1020-1025.

An aliquot of the test sample (0.2 mg of protein) was added to the testtubes containing 0.15M potassium phosphate buffer, pH 7.4. Proteins wereprecipitated by adding a 20% TCA solution. After the samples werecentrifuged at 1000 g for 15 minutes, a 0.1M solution of 2,4-DNPH in 2 MHCl was added to the precipitate of denatured proteins. After an hourincubation at room temperature, the precipitate was washed three timeswith ethanol:ethyl acetate (1:1) mixture to remove lipids and 2,4-DNPH,which were not bound to carbonyls. The so washed precipitate was driedand dissolved in 8M urea in a boiling water bath for 10 minutes.

To determine the aldehyde and ketone products of the oxidativemodification of proteins, the optical density was measured at awavelength of 356 nm and 370 nm, respectively. The obtained values wererecalculated using an appropriate molar extinction coefficient.

Determination of the Levels of Serotonin and Tryptophan in the Brain andDuodenal Homogenates and Serum

Aliquots of serum and tissue homogenates were mixed with 0.4M perchloricacid at the ratio of 1:5 to precipitate proteins. The samples wereincubated at 4° C. for 60 minutes and then centrifuged at 800 g for 5min in a refrigerated centrifuge at 4° C. After the phase separation,the supernatant was collected and pH was adjusted to 5-6 with 2 M KOH.The samples were reprecipitated by centrifugation. The supernatant wasapplied to a KM-Sepharose column previously equilibrated with 0.01Msodium phosphate buffer, pH 6.2. The bound material was eluted at roomtemperature using buffer 1 (0.01M sodium phosphate buffer, pH 6.2) andbuffer 2 (0.03M sodium phosphate buffer, pH 6.2). Tryptophan was elutedusing buffer 1, and serotonin was eluted using buffer 2.

Tryptophan levels were measured with a spectrofluorometer at anexcitation wavelength of 295 nm and an absorption wavelength of 550 nm,versus a blank sample which, instead of the test sample, contained acorresponding volume of distilled water.

Serotonin levels were measured with a spectrofluorometer at anexcitation wavelength of 359 nm and an absorption wavelength of 485 nm,versus a blank sample which, instead of the test sample, contained acorresponding volume of distilled water. Gaitonde M. K.//Biochem.S.-1974.-Vol. 139.-P. 625-631. Maksymenko E. G., Savchenko V. N.//Visnykof V. N. Karazin Kharkiv Nat. University. Medicine.-2000.-1, No. 494.-P.40-43. H. Weissbach et al.,/J Biol Chem//-1957.-Vol. 230, No 2.-P.865-71.

Determination of Tryptophan Hydroxylase Activity in Brain and DuodenalHomogenates

Tryptophan hydroxylase activity was determined as described by Donald M.Kuhn, at al., Biochemistry.-1980.-Vol. 77.-P. 4688-4691. Tissuehomogenates were thawed at room temperature and centrifuged at 12000 gfor 30 min. The supernatant was used in further studies.

An incubation medium was prepared in Ependorf microcentrifuge tubes thatcontained 500 mM Tris-HCl, pH 7.4, 20 mM dithiotrietol, 1 mM CaCl₂, 4 mML-tryptophan and 50 μg catalase, to which an aliquot of the supernatantwas then added. The samples were incubated in a thermostat at 37° C. for15 min. The reaction was stopped by precipitating proteins with 6MHClO₄. To separate the precipitated proteins, the samples werecentrifuged at 600 g for 5 minutes.

The optical density of the samples was measured at 295/540 nm with aspectrofluorometer. A blank sample containing the incubation medium anddistilled water was used as a control.

Determination of indolamine-2,3-dioxigenase Activity in Brain andDuodenal Homogenates

Indolamine-2,3-dioxigenase activity was determined as described by Y.Kudo, C. A. R. Boyd, I. L. Sargent et al.//Mol. Humanreproduction.-2000.-Vol. 6, N 4.-P. 369-374. Tissue homogenates werethawed at room temperature and centrifuged at 12000 g for 30 min. Thesupernatant was used in further studies.

An incubation medium was prepared in Ependorf microcentrifuge tubes thatcontained 100 mM potassium-phosphate buffer, pH 7.5, 5 mM L-tryptophan,10 mM ascorbate, 0.2 mM methylene blue, 50 μg catalase, to which analiquot of the supernatant was then added. The samples were incubated ina thermostat at 37° C. for 30 min. The reaction was stopped byprecipitating proteins with 10% trichloroacetic acid. To separate theprecipitated proteins, the samples were centrifuged at 600 g for 5minutes. Then 1M Tris-HCl, pH 7.0 was added to the aliquot of thesupernatant.

The optical density of the samples was measured at 360 nm using aspectrofluorometer versus a blank sample that contained the incubationmedium and distilled water.

Determination of Serum Monoamine Oxidase Activity

Serum monoamine oxidase activity was determined using a method describedby Balakleevsky A. I.//Lab. business.-1976.-3.-P. 151-152. The methodconsists in the formation of benzaldehyde from benzylamine hydrochlorideunder the action of MAO. Benzaldehyde interacts with2,3-dinitrophenylhydrazine and forms insoluble hydrazone which can beprecipitated by centrifugation. The hydrazone precipitate, in turn,forms a stable compound of raspberry color in an alkaline medium, thecontent of which can be determined spectrophotometrically.

An incubation medium was prepared in Ependorf microcentrifuge tubes thatcontained 0.2M phosphate buffer, pH 7.4, distilled water, and a 1%solution of benzylamine hydrochloride. A blank sample did not containbenzylamine hydrochloride. The reaction was started by addition of analiquot of serum. The samples were incubated in a thermostat at 37° C.for 3 hours. The reaction was stopped by precipitating proteins with 10%trichloroacetic acid. To separate the precipitated proteins, the sampleswere centrifuged at 600 g for 5 minutes. A 0.1% solution of2,3-dinitrophenylhydrazine prepared in 2M HCl was added to the resultingsupernatant. The samples were stirred and incubated for 25 minutes atroom temperature. After that, hydrazone was precipitated bycentrifugation of the samples at 600 g for 25 minutes. 3M NaOH and 96%ethanol were sequentially added to the hydrazone precipitate, and thedevelopment of a raspberry color was observed.

The optical density of the samples was measured using aspectrofluorometer with excitation at 460 nm versus ethanol.

Morpho-Functional Analysis of Pancreatic and Liver Tissues

At the end of the experiment, the animals were euthanized bydecapitation. Prepared liver and pancreas 0.5-0.5 cm in size wereimmediately placed in a fixative. The organs were fixed in a 4% solutionof paraformaldehyde at a temperature of 25° C. for 72 hours. Afterfixation, the pieces were rinsed in tap water. Then the material wasdehydrated. This was achieved by passing the pieces of organs throughincreasing concentrations of alcohol (70%>80%>90%>96%) leaving them fora day in each concentration. Finally, once the water was replaced by 96%alcohol, the material was placed in dioxane for 15 minutes, and then inxylene for 15 minutes. After complete clearing, the material was placedin a paraffin bath (paraffin and xylene mixture at a 1:1 ratio) in athermostat for 30 minutes at 37° C. The material was then submerged intwo changes of paraffin (30-35 minutes) in a thermostat at 56° C., andparaffin blocks were produced.

A series of 5 μm thick histological sections of tissue were cut using anMS-2 sliding microtome and placed on glass slides treated with a 1:1mixture of protein and glycerol.

Dried preparations were stained with hematoxylin and eosin. Prior tostaining, the sections were dewaxed in 2 changes of xylene for 5 minutesand passed through decreasing strengths of alcohol (96%>90%>80%>70% for3 minutes in each) and finally distilled water for 5 minutes. Thesections were stained with Bomer's hematoxylin for 1.5 minutes thenwashed in running water for 15-20 minutes and stained with eosin for 1minute. Once stained, the sections were dehydrated once again in 70% and96% alcohols (30 seconds in each) and cleared in dioxane and xylene for2.5 minutes. The stained sections were enclosed in Canada Balsam andcovered with coverslips. The nuclei of cells had a blue-violet color andthe cytoplasm was pink.

To carry out a histochemical reaction to determine the level of liverfibrosis, Van Gieson's picro-fuchsin method of staining was used. To dothis, the sections were first soaked with water and then re-stained withBömer's hematoxylin for 3-4 minutes. The sections were then rinsed indistilled water and stained with Van Gierson's picro-fuchsin for 3minutes. Once stained, the sections were rinsed in distilled water,dehydrated in 96% alcohol, cleared in dioxane and xylene, and enclosedin a balsam under a coverslip. As a result, hepatocyte nuclei had a darkbrown color, collagen fibers were red and the cytoplasm was yellow. Allparameters were measured using ImageJ software.

Determination of Protein Concentration

The protein concentration was measured using the Bradford protein assay.Bradford M M. Anal Biochem. 1976; 86: 193-200. To measure the proteinconcentration, 10% NaOH, distilled water, and Bradford reagent wereadded to the sample. Bradford reagent was prepared by mixing the initialsolution (95% ethanol, 85% H₃PO₄ and Coomassie Brilliant Blue dye) with95% ethanol and 85% H₃PO₄, and adjusting the resulting mixture to thedesired volume with distilled water.

The absorbance was measured spectrophotometrically at 595 nm versus acontrol sample that contained distilled water instead of the testsample. The protein concentration was determined using a calibrationcurve and was expressed in mg/ml.

Statistical Processing of the Results

Statistical processing of the obtained results was carried out using themethods of variation statistics and correlation analysis using OrginLabOrgin® Pro 9.1 and StatSoft STaStica® 10 software. Brandt Z. Statisticalmethods for observations.-M.: Mir, 1975.-312 p. The hypothesis of normaldistribution of samples was tested using the Shapiro-Wilk test. If asample met the criteria of normal distribution, significance ofdifferences between samples was determined using the Student's t-test.If a sample did not meet the criteria of normal distribution,significance of differences between samples was determined using theMann-Whitney U test. Differences were considered statisticallysignificant when p<0.05.

Results and Discussion

Biochemical and Anthropometric Effects of Zn-64 Stable Isotope inAspartate Form in Animals Models of Obesity

According to modern concepts, adipose tissue, producing a wide range ofbiologically active substances, is actively involved in the pathogenesisof obesity. Therefore, overweight occurring due to an increase in fatdeposits is considered not only as a consequence of metabolic disordersduring the development of obesity but also as an important factor thatprovokes and greatly complicates the course of the disease, contributingto the development of a number of obesity-related disorders.

To assess the effects of Zn-64 stable isotope in aspartate form on thedevelopment of obesity induced by the consumption of high-fat foods,some anthropometric values were evaluated in animal models of obesityand animals treated with Zn-64 stable isotope in aspartate form.

A characteristic morphological sign of the development of obesity is asignificant increase in body weight due to accumulation andredistribution of adipose tissue. To confirm the development of obesity,the body mass index (BMI) or Quetelet index, which is body weight inkilograms divided by the square of the height in meters, was firstdetermined. Novelli E., Diniz Y., Galhardi C. Anthropometricalparameters and markers of obesity in rats//Laboratory Animals.-2007.-No41.-P. 111-119. BMI makes it possible to assess the body massrelationship to height and thereby indirectly assess whether the mass isinsufficient, normal or excessive. In addition, BMI is used as anintegral value, which allows us to characterize body composition and adegree of fat deposits because the character of distribution of adiposetissue in the body determines the risk of developing metaboliccomplications associated with obesity, which must be considered whenexamining obese patients. BMI is not only used to classify obesity butalso to determine risks of developing obesity-related diseases.

The data obtained during the experiment show (Table 8) that on the10^(th) week of the experiment, the mean body mass index of controlanimals was 0.60 g/cm² which value was within a reference range foranimals of this age group. Novelli E., Diniz Y., Galhardi C.Anthropometrical parameters and markers of obesity in rats//LaboratoryAnimals.-2007.-No 41.-P. 111-119. BMI of animals eating high-fat dietwas 1.14 times higher than BMI of animals of the control group (0.71g/cm²). It should be noted that the body mass index of rats receivingZn-64 stable isotope in aspartate form during the experiment was lowerthan that of obese animals but slightly higher than the control values(0.65 g/cm²). The obtained result indicates that Zn-64 stable isotope inaspartate form has a positive effect on the general metabolic status ofanimals and lays the groundwork for further studies aimed at finding outmechanisms of effects of Zn-64 stable isotope in aspartate form onobesity.

Since BMI is calculated based on weight, a decrease in BMI value may bedirectly related to the lower weight of animals that received Zn-64stable isotope in aspartate form. Therefore, it was further investigatedwhether administration of Zn-64 stable isotope in aspartate formaffected the weight and weight gain of animal models of obesity. Thedata obtained in the experiment show (FIG. 5) that the dynamics ofweight gain by animals of the experimental groups differedsignificantly. Thus, animals that were maintained on a high-fat diet andreceived Zn-64 stable isotope in aspartate form gained less weight thananimals that were only fed a high-fat diet. Particularly noticeabledifference in the weight gain of animals of both groups was observedstarting from the 4^(th) week of the experiment. An increase in bodyweight of animals eating a high-fat diet reached 103% by the end of theexperiment, while animals that received intragastric injections of Zn-64stable isotope in aspartate form gained almost as much weight as animalsin the control group (62%).

It is known that the development of obesity, due to disruption of thecoordinated work of a number of neurotransmitter and hormonal systems ofthe body, leads to disturbances at the level of appetite control andregulation of a feeling of satiety, which promotes excessive food intakeand is often accompanied by the development of hyperphagia, a state ofan abnormally great desire for food energy equivalent of which exceedsthe energy needs of the body. L. Zhou, G. Sutton, J. Rochford.//CellMetabolism.-2007.-Vol. 6, No 5.-P. 398-405.

To figure out possible mechanisms of the effect of decrease in the bodyweight of animals receiving Zn-64 stable isotope in aspartate form, theamount of food that animals consumed was analyzed. Table 8.

Comparing the data calculated for all experimental groups, it can beseen that there are no particular differences in the amount of food theanimals ate on average per day. Thus, animals of the control group andthe group of obese animals consumed about 35 g of food per day. But hereit should be noted that animals in the control group were maintained ona standard diet while animals in the group of diet-induced obesitymodels consumed specially prepared high-fat diet the caloric content ofwhich was significantly higher.

TABLE 8 Some anthropometric values, the amount and caloric content food(M ± m, n = 10) Experimental groups C C + zinc DIO DIO + zinc BMI(g/cm²) 0.60 0.59 0.71 0.65 Weight gain as of the end of the 59 59 10362 experiment (%) Amount of food consumed (g/day) 34 32 35 29 Caloriccontent of food (kJ/day) 525 490 1001 823 C-control; C + zinc-control onthe background of administration of Zn-64 stable isotope in aspartateform; DIO-diet induced obesity; DIO + zinc-diet induced obesity on thebackground of administration of Zn-64 stable isotope in aspartate form.

Analysis of the results obtained, with due consideration of the caloriccontent of food consumed by animals, shows a significant difference invalues. Despite the same amount of food eaten by animals of the controlgroup and the group of diet-induced obesity models, the caloric contentof food differed almost twice. A result obtained from the groupadministered with Zn-64 stable isotope in aspartate form seems quiteinteresting. Thus, animals of the control group and the group ofdiet-induced obesity models ate smaller amounts of standard and high-fatdiets, respectively.

The dynamics of caloric content of food consumed by animals during 10weeks of the experiment is shown in FIG. 6.

The data obtained suggest that Zn-64 stable isotope in aspartate formhas an effect on the feeling of satiety because, having free access tofood, animals injected with Zn-64 stable isotope in aspartate formconsumed significantly less food compared to animals that weremaintained only on a high-fat diet. A decrease in the amount of foodconsumed and, consequently, insignificant weight gain in animals thatreceived Zn-64 stable isotope in aspartate form compared with animals inthe group of diet-induced models can be explained by both direct andindirect effects of zinc on energy homeostasis.

Thus, summarizing the results of this phase of the study, administeringZn-64 stable isotope in aspartate form caused a decrease in the amountof food consumed per day, which, accordingly, was accompanied by a lesspronounced weight gain in animals and normalization of their body massindex in comparison with similar values in obese animals that were nottreated with Zn-64 stable isotope in aspartate form.

An early detection of Zn deficiency states is of paramount importance toprevent the onset and development of metabolic disorders. A laboratorysign of zinc deficiency is a decrease in its levels in the blood plasma(serum), but plasma zinc levels are labile and are influenced by manyfactors.

There are other approaches to determining zinc status. They, inparticular, are based on the measurement of concentration ofzinc-dependent proteins and, first of all, such enzymes as carbonicanhydrase, superoxide dismutase, lactate dehydrogenase and alkalinephosphatase, as well as metallothionein, a serum retinol-bindingprotein, in plasma (serum). One of the earliest markers of zincdeficiency is a decrease in the activity of serum alkaline phosphataseand carbonic anhydrase. As a result, stress ulcers caused by a highcontent of such zinc-containing enzyme as carbonic anhydrase in themucosa are developed in the gastrointestinal tract. Therefore, toindirectly determine whether the development of obesity is accompaniedby changes in zinc status, alkaline phosphatase activity in the bloodserum of obese animals and animals treated with Zn-64 stable isotope inaspartate form was studied.

A significant decrease in the activity of this enzyme in animalsmaintained on a high-fat diet was observed (Table 9). Thus, the enzymeactivity in these animals was 1.5 times lower than in animals of thecontrol group. In animals treated with Zn-64 stable isotope in aspartateform, alkaline phosphatase activity was higher than both in the group ofdiet-induced obesity models and in the control group.

Thus, the obtained results indirectly confirm zinc deficiency in animalmodels of obesity and normal serum zinc levels in animals receivingZn-64 stable isotope in aspartate form.

The gastrointestinal tract maintains whole-body zinc homeostasis. Thereare no true depots of this trace element in the human body. Zincabsorbed from the intestine enters the bloodstream. The whole bloodcontains about 7-8 mg/L of zinc, what is more, about ⅔ of this amount istransported by red blood cells. In plasma, about 80% of zinc is bound toalbumin and the other 20% is bound to β2-macroglobulin and transferrin.Published data confirm dependence of the levels of this trace element onthe concentration of albumin in the blood plasma. Brown, K. H.International Zinc Nutrition Consultative Group (IZiNCG) technicaldocument #1. Assessment of the risk of zinc deficiency in populationsand options for its control/K. H. Brown, J. A. Rivera, Z. Bhutta [etal.]//Food Nutr. Bull.-2004.-Vol. 25.-P. 99-203.

Therefore, albumin levels in untreated animal models of obesity andobese animals treated with Zn-64 stable isotope in aspartate form wasfurther investigated. The data available from the experiment show thatthe pathogenesis of obesity is accompanied by a decrease in the serumalbumin levels in animals. At the same time, administration of Zn-64stable isotope in aspartate form had no effect on albumin values whichremained similar to values in untreated obese animals (Table 9).

TABLE 9 Serum biochemistry of experimental animals (M ± m, n = 10)groups parameters Control DIO DIO + zinc Alkaline phosphatase  74.3 ±12.1  37.2 ± 15.4*  87.6 ± 18.7# activity, RU Albumin levels, RU 219.2 ±14.6 168.8 ± 16.8* 166.2 ± 15.8* Triglycerides, g/L  2.55 ± 0.20  4.39 ±0.73*  2.79 ± 0.30# Cholesterol, mmol/L  2.42 ± 0.19  5.76 ± 0.87*  2.83± 0.23# Free fatty acids, mg/L 23.60 ± 4.67 74.50 ± 9.23* 31.62 ± 7.92#*the difference is significant versus the control group of animals; #thedifference is significant versus the group of animal models of obesityC-control; C + zinc-control on the background of administration of Zn-64stable isotope in aspartate form; DIO-diet induced obesity; DIO +zinc-diet induced obesity on the background of administration of Zn-64stable isotope in aspartate form.

Considering that albumin acts as the major transport protein for zinc, adecrease in its concentration will cause a disruption in timely deliveryof zinc to the organs, including liver, where synthesis of the mainzinc-containing proteins occurs.

In general, this result is fully consistent with a decrease in alkalinephosphatase activity established above.

It has also been found that Zn-64 stable isotope in aspartate form has apositive effect on lipid metabolism in the body. A decrease in thelevels of triglycerides, cholesterol and free fatty acids in the serumof animals that were fed a high-fat diet and treated with Zn-64 stableisotope in aspartate form was almost at the same level as in the controlgroup of animals.

The pathogenesis of obesity due to metabolic disorders, mostly due todisturbances in hydrocarbon metabolism, is usually accompanied by anincrease in glucose levels, which, if remain high for a long time,launch a number of pathological processes and become a significantfactor that induces the development of insulin resistance and diabetes.Today, it is a proven fact that there is a relation between changes inthe levels of trace elements, zinc in particular, and the onset ofprediabetes and, in the absence of proper pharmacological correction,the development of diabetes. According to research results,concentrations of most trace elements in the body are constant, but inthe case of zinc, a decrease in its levels in the blood serum of womenwith pre-diabetes has been shown. It is known that this element plays animportant role in insulin synthesis in beta cells in the pancreas, andit also enhances susceptibility of tissues to this hormone. Chausmer, A.B. Zinc, insulin and diabetes. J. Am. Coll. Nutr. 1998, 17, 109-115.

In view of the above, the effects of Zn-64 stable isotope in aspartateform on glucose concentrations and insulin levels in the blood serum ofanimals eating a high-fat diet were investigated.

Based on the literature, fasting blood glucose levels within the rangeof 3.5-5.5 mmol/L are considered normal. An increase in this value overa certain time period to 7.0 mmol/L and above is regarded as a state ofhyperglycemia and may be a predictor of the development of diabetesmellitus.

Serum glucose levels in animals from the control group and animals fromthe control group that received Zn-64 stable isotope in aspartate formwere within the reference values (Table 10). The development of obesitywas accompanied by a slight increase in the glucose levels which werenormalized by the administration of Zn-64 stable isotope in aspartateform.

The effect of Zn-64 stable isotope in aspartate form on glucose levelsmay be directly related to its ability to stimulate the movement of theglucose transporter from inner cell compartments to adipocyte membranes,which contributes to the enhancement of intracellular glucose transport.It has also been found that Zn-64 stable isotope in aspartate formincreases tyrosine phosphorylation of the insulin receptor β-subunit,thus improving glucose transport in the absence of insulin. The dataindicate that Zn-64 stable isotope in aspartate form can act as aninhibitor of tyrosine-1B-phosphatase, an enzyme involved in thesuppression of insulin signaling.

TABLE 10 Serum glucose concentrations and insulin levels in experimentalanimals (M ± m, n = 10) Experimental Insulin levels, Glucose levels,groups RU mmol/L C 0.133 ± 0.024 4.4 ± 0.3 C + zinc 0.145 ± 0.013 4.7 ±0.2 DIO 0.216 ± 0.035* 7.1 ± 0.1* DIO + zinc 0.149 ± 0.018# 4.9 ± 0.2#*the difference is significant versus the control group of animals; #thedifference is significant versus the group of animal models of obesityC-control; C + zinc-control on the background of administration of Zn-64stable isotope in aspartate form; DIO-diet induced obesity; DIO +zinc-diet induced obesity on the background of administration of Zn-64stable isotope in aspartate form.

An increase in glucose concentrations in obesity may be a consequence ofa decrease in insulin secretion in βcells in the pancreas or itsinadequate utilization by the tissues of the body. High glucose levelsin the blood and other body fluids causes an increase in osmoticpressure resulting in the development of osmotic diuresis (increasedloss of water and salts through the kidneys), which leads to dehydrationof the body and deficiencies in sodium, potassium, calcium and magnesiumcations, chlorine anions, phosphates and hydrocarbonates. In addition,elevated glucose levels cause non-enzymatic glycosylation of proteinsand lipids, the intensity of which is directly proportional to glucoseconcentrations. As a consequence of this, the functions of many vitalproteins are impaired resulting in various pathological changes in thebody. Skybchyk V.//Ukrainian medical newspaper.-2006.-No 6.-P. 61-68.Campos.//Postgraduate Medicine.-2012.-No 126.-P. 90-97.

Considering changes in the serum glucose levels in animals, the nextphase of this study was to determine the levels of insulin. In addition,a serum insulin level is an important parameter in diagnosing thedevelopment of insulin resistance and prediabetes. In obesity andmetabolic syndrome, hyperinsulinemia is often caused by excessiveproduction and secretion of insulin in β-cells in the pancreas, which isa compensatory response to a decrease in the sensitivity of peripheraltissues to insulin action. However, in later stages of the developmentof type 2 diabetes mellitus, serum insulin levels are significantlyreduced, which is directly related to impaired ability of β-cells toproduce insulin, impaired proinsulin processing and secretion of matureinsulin, as well as a reduction in the number of secreting cells anddeposition of amyloid in the islets of Langerhans. At the same time, thedeveloping β-cell dysfunction causes further progression of diabetesmellitus. Boden.//Diabetes.-1997.-Vol. 46, No 3.-P. 3-10. Robertson R.P. et al.//Diabetes Mellitus.-2000.-P. 125-132.

An increase in the serum insulin levels was found in obese animals and anormalizing effect of Zn-64 stable isotope in aspartate form on thestudied parameter in the group of animals maintained on a high-fat dietand receiving Zn-64 stable isotope in aspartate form. It should beemphasized that administering Zn-64 stable isotope in aspartate form toanimals of the control group caused a slight increase in the insulinlevels.

The effect of Zn-64 stable isotope in aspartate form on insulin levelsmay be associated with the direct involvement of this trace element inthe processes of synthesis, deposition and release of insulin fromβ-cells in the islets of Langerhans as well as its ability to inhibitthe action of insulinase. It is known that zinc is involved in theformation of hexameric proinsulin and contributes to the crystallizationof insulin. It has been proven that zinc ions contribute toincorporation of insulin into the transport complex which ensures itsdelivery to target cells. Another possible mechanism was established in1980 by Caulston and Dandona, who demonstrated that zinc has a powerfuland stimulating, independent and complementary to insulin action, effecton lipogenesis in rat adipocytes. This discovery confirmed theinvolvement of zinc in controlling the effects of insulin, as thiscation is secreted along with insulin in response to high glucoselevels.

In addition, zinc plays an important role in protecting insulin andpancreatic beta cells from free radicals, as it is a structuralcomponent of antioxidant enzymes, such as superoxide dismutase, and acompetitor to redox metals, such as iron. Zinc stimulates the expressionof metallothioneins in the pancreatic cells known to be involved in theneutralization of a number of active oxygen metabolites and be able toprevent the destruction of beta cells.

Given the importance of maintaining physiological levels of zinc in thebody to ensure the synthesis and secretion of insulin as well as itsimportant role in the pancreas function, the effects of Zn-64 stableisotope in aspartate form on the overall morphofunctional properties ofpancreas were further investigated.

Effects of Zn-64 Stable Isotope in Aspartate Form on theMorphofunctional Properties of Pancreas and Liver of Animal Models ofObesity

The pancreas is a mixed gland, having both exocrine and endocrinefunction. The bulk of pancreas is composed of exocrine cells arranged inacini. Secretions from acini flow out of the pancreas throughintercalated, intralobular and interlobular ducts and the mainpancreatic duct. Clusters of exocrine cells, the acini, in animals fromthe control group (FIG. 7A-FIG. 7F) have a typical structure: cytoplasmin the apical pole is granular and brightly acidophilic, while the basalpole contains nuclei which are strongly basophilic.

In the animal models of DIO (obesity group) (FIG. 7A-FIG. 7F), therewere acini with not very noticeably eosinophilic apical cytoplasm (FIG.7A-FIG. 7F, arrows), which may be due to the accumulation of lipidinclusions, pancreatic fatty degeneration.

Administration of Zn-64 stable isotope in aspartate form to rats thatwere fed a standard diet did not change morphology of exocrine cells(FIG. 8A-FIG. 8F). After administration of Zn-64 stable isotope inaspartate form to rats having obesity (FIG. 8A-FIG. 8F), no fattydegeneration was found.

The endocrine part of the pancreas is composed of diffusely locatedislets of Langerhans. A morphometric study of the functional state ofthe endocrine pancreas during the development of induced obesity showedclear differences between the values obtained from all groups (FIG. 9).A cross-sectional surface area of the islets of Langerhans wassignificantly reduced in the animals from the obesity group (by 60%),which indicates a significant decrease in the functional activity oftheir endocrine pancreas. After administration of Zn-64 stable isotopein aspartate form to rats having obesity, the cross-sectional surfacearea of the islets of Langerhans increased by 43% compared with theobesity group, but still did not reach the control level (lower than thecontrol value by 29%). Administration of Zn-64 stable isotope inaspartate form to rats that ate a standard diet caused a noticeabledecrease in this value by 39% compared with the control group.

Since there is a direct relationship between the morphological andfunctional indicators of the state of pancreas, the data obtained showthat the hormone-synthesizing activity of the pancreas in diet-inducedobesity rat models is significantly reduced but it increases markedlywith the administration of Zn-64 stable isotope in aspartate form,though it does not fully restore to the levels observed in the controlgroup. In addition, an improvement in the state of the exocrine part ofthe pancreas after administration of the test substance against thebackground of the development of obesity was recorded, which isevidenced by disappearance of fatty degeneration, without a noticeableeffect on exocrine cells in rats fed a standard diet.

The liver of control rats (FIG. 10A-FIG. 10D) has a classical lobularorganization with a central vein running along the axis of each lobule.Hepatocytes of polygonal morphology with clearly defined nuclei, whichcontain several nucleoli each, are arranged into ordered hepatic cordsspreading from the central vein. Binuclear hepatocytes are also found.

In animals from the obesity group (FIG. 10A-FIG. 10D), the hepatocyteshape changes from polygonal to rounded due to the deposition of lipidinclusions, which is a sign of fatty degeneration of the liver. Thestructure of the hepatic cords is disarranged and the number ofbinucleate cells in the field of view is reduced.

As a result of administration of Zn-64 stable isotope in aspartate formto rats having obesity (FIG. 11A-FIG. 11D) the structure of hepaticcords was restored, most hepatocytes of polygonal morphology showed nosigns of fatty degeneration, binucleate cells were often found.Administration of Zn-64 stable isotope in aspartate form to rats eatinga standard diet (FIG. 11A-FIG. 11D) did not cause any changes in themorphology of hepatocytes and the structure of hepatic lobules.

With the development of diet-induced obesity, significant morphometricchanges in hepatocytes occurred (FIG. 12A-FIG. 12C). Thus, in animalsfrom the obesity group, the nucleus area decreased by 25% (which isevidence of a decrease in transcriptional activity of the nucleus, alsoconfirmed by its dark color and homogeneous structure with no nucleoliin the field of view), but the area of hepatocytes increased by 48% dueto the deposit of a large number of lipid inclusions. At the same time,the nucleus-to-cytoplasm ratio significantly reduced (by 45%), a lowlevel of which indicates that the functional activity of the cellsdecreased.

Administration of the test substance to obese rats improved theirmorphometric parameters. Thus, the area of hepatocytes in animalstreated with Zn-64 stable isotope in aspartate form decreased by 41%versus untreated animal models of obesity (only a 13% increase comparedto the control values, which is a sign of a decrease in the depositionof lipid inclusions by hepatocytes) and their nucleus-to-cytoplasm ratioincreased by 31% compared to the obesity group (a decrease by 30%compared to the control group).

However, the nucleus area is reduced versus the control values by 35%(i.e. decreased in relation to the values in the obesity group by 14%,which may be the result of a combined effect of two factors—a high-fatdiet and Zn-64 stable isotope in aspartate form—on the nuclearactivity).

The effect of Zn-64 stable isotope in aspartate form on the morphometricparameters in rats that were maintained on a standard diet consisted inthe reduction of the nucleus area by 26%, the area of hepatocytes by 12%and the nucleus-to-cytoplasm ratio by 17%.

Liver fibrosis is characterized by excessive growth of connectivetissue, an increased synthesis and deposition of collagen in theextracellular substance. In the samples taken from animals from thecontrol group (FIG. 13A-FIG. 13D), most of the collagen fibers were inthe region of triads formed by small interlobular vessels.

Samples taken from the animals from the obesity group (FIG. 13A-FIG.13D) showed a noticeable increase in the number of collagen fibers inthe triad region formed, as in the control group, by small perilobularcapillary plexuses and larger interlobular vessels.

Samples taken from obese rats injected with Zn-64 stable isotope inaspartate form (FIG. 14) show similar levels of deposition of collagenfibers in the indicated places when compared to samples from theuntreated obesity group. Administration of the test substance to ratsthat were fed a standard diet (FIG. 14A-FIG. 14D) caused no significantchanges in the amount of collagen fibers in the perilobular andinterlobular capillary plexuses.

Analysis of the area occupied by collagen fibers (FIG. 15) showssignificant changes with the development of induced obesity. The area ofcollagen fiber deposits in the obesity group and in the group treatedwith Zn-64 stable isotope in aspartate form increased by a factor of6.25 and by a factor of 6, respectively, versus the control group. Nosignificant differences between the untreated obesity group and theobesity group treated with Zn-64 stable isotope in aspartate form werefound. Administration of the test substance to rats eating a standarddiet caused a significant increase in the area of deposition of collagenfibers (2-fold).

Thus, summarizing the results obtained, it can be said that Zn-64 stableisotope in aspartate form has a positive effect on the morphofunctionalproperties of the pancreas and liver of animal models of obesity.

Effects of Zn-64 Stable Isotope in Aspartate Form on the SerotoninergicSystem in Animal Models of Obesity

Despite a proven fact that the development of obesity is primarily theresult of an increased caloric intake and inadequate energy expenditure,a search for new pathogenetic mechanisms of weight gain is stillrelevant.

According to modern concepts, obesity, regardless of its etiology, leadsto a disruption in the central regulatory mechanisms that affectbehavioral responses, eating behavior, in particular. In thehypothalamus, mainly in the region of its paraventricular nuclei andlateral perifornical area, an integration of a multitude of impulsescoming from the cerebral cortex and subcortical structures to thesympathetic and parasympathetic nervous systems takes place. Adisruption in any link of this complex regulatory cascade can lead tochanges in food intake, fat deposition and mobilization and, ultimately,to the development of obesity.

Important neurotransmitters involved in the regulation of eatingbehavior, appetite in particular, and affecting the feeling of satiety,include a number of biogenic amines, among which serotonin plays adecisive role. C. Portas et al., Progress in Neurobiology.-2000.-Vol.60, No 1.-P. 13-35.

According to the theory of neurochemical imbalance of the central andperipheral nervous systems, overeating is a compensatory mechanism forobtaining pleasure due to insufficient production or sensitivity ofneurotransmitters.

It is the central serotonergic system that is fundamental in regulatingthe feeling of hunger and satiety. Experiments demonstrated that anincrease in serotonergic transmission in the brain caused a decrease infood intake. Injections of 5-HT into the paraventricular nucleus of therat hypothalamus led to satiation of the animal, whereas, with foodconsumption, an increased yield of serotonin in the lateral hypothalamuswas noted. These two sections of the hypothalamus are believed toperform the opposite function in regulating appetite: insufficientinhibition of serotonergic transmission in the lateral hypothalamus maybe a cause of excessive food intake during obesity, and enhanced releaseof serotonin in the paraventricular nucleus of the hypothalamus maycontribute to stress induced hypophagia.

The brain and intestines are the main organs producing serotonin inanimals. Since serotonin does not cross the blood-brain barrier, theserotonin synthesis system is divided into central and peripheral, whichoperate separately from each other. A small amount of the hormone is inthe plasma.

Several enzyme systems are involved in metabolic transformations ofserotonin. These systems include enzymes for the serotonin synthesis anddegradation, as well as an enzyme which determines how much tryptophanenters the kynurenine pathway. A. Meneses, G. Liy-Salmeron.//AnnualReview of Neuroscience.-2012.-No 23.-P. 543-553. M. Donovan, L.Tecott.//Frontiers in Neuroscience.-2013.-No 7.-doi:10.3389/fnins.2013.00036.

Tryptophan, an essential amino acid naturally produced by the body, isthe immediate precursor of serotonin. Tryptophan is transported from theextracellular fluid to serotonergic neurons by a non-specific membranetransporter, which is believed to be involved in the transport of otherneutral amino acids (valine, leucine, isoleucine). Therefore, levels oftryptophan in neurons and the intensity of its transport depend not onlyon the concentration of tryptophan, but also on the ratio ofconcentrations of competing neutral amino acids and the concentration oftryptophan.

Serotonin is synthesized through a two-step process catalyzed by twoenzymatic systems. First, as a result of hydroxylation in the fifthposition of the indole ring, tryptophan is converted to5-hydroxytryptophan, a direct precursor of serotonin synthesis. Thereaction of tryptophan hydroxylation is catalyzed by the rate-limitingenzyme tryptophan hydroxylase (tryptophan-5-monooxygenase, EC 1.14.16.4)in the presence of both molecular oxygen and pterin(tetrahydrobiopterin) as coenzymes. The rate of tryptophan hydroxylationdirectly depends on the availability of the substrate. The second stepin the synthesis of serotonin is decarboxylation catalyzed by DOPAdecarboxylase (also known as aromatic L-amino acid decarboxylase, EC4.1.1.28).

In addition to participation of these enzymes in the synthesis ofserotonin and other biologically active amines, both enzymes areactively involved in the regulation of circadian rhythms, boneremodeling, cell differentiation processes, immune response mechanismsand inflammation.

Inactivation of serotonin occurs by enzymatic degradation, which ismainly served by monoamine oxidase (EC 1.4.3.4). Under the action ofMAO, serotonin is converted to 5-hydroxyindaldehyde, which, in turn, mayreversibly convert to 5-hydroxytryptophol under the action of alcoholdehydrogenase. Under the action of acetaldehyde dehydrogenase,5-hydroxyindaldehyde is irreversibly converted into 5-hydroxyindoleacetic acid, which is then excreted with urine and feces. Sandler M. etal, Clinical Pathology.-1981.-No 34.-P. 292-302. S. Nilsson, N. et al,Acta medica Scandinavica.-1968.-No 184.-P. 105-108.

When released, serotonin influences various biological processes bybinding to serotonin receptors (HTR). Its action is then terminated byuptake in cells through the serotonin transporter (SERT, Slc6a4).

In addition to oxidative deamination of serotonin, other pathways ofserotonin metabolism are possible, for example, the pathways ofacetylation and glucuronic acid and sulphate ester conjugations. Thereis a pathway for serotonin metabolism, which is accompanied by theformation of melatonin.

Medical studies have shown that a disorder in metabolic transformationsof serotonin is often not only a consequence, but also one of the mainfactors that trigger the development of overweight and obesity.Molecular and biochemical disorders at the level of different phases ofserotonin metabolism, its transport through cell membranes anddeposition mechanisms leading to changes in serotonin concentrationsboth in the central nervous system and in the periphery may be one ofthe defining pathological bases for the formation of immuneneuroendocrine imbalance and maintaining proinflammatory reactions.Imbalance of the serotoninergic system is an underlying cause of thedevelopment of a number of pathological conditions of the body andmental disorders, including schizophrenia, various psychoses, depressionand anxiety. Depressive states are often accompanied by an increasedappetite, which causes excessive food intake, especiallycarbohydrate-rich foods.

Recent research data have shown that zinc can modulate serotonergicfunction via the 5-HT1A receptors (5-HT1AR); however, the exactmechanisms of its action are unknown. Considering the above, as well asthe importance of zinc for proper functioning of the neurohormonalsystem of the brain, its involvement in the metabolism of tryptophan, akey molecule of serotonin synthesis, and a modulating effect of zinc onthe serotonergic system, the main indicators that would allow a generalassessment of serotonin metabolism in experimental animals were furtheranalyzed. For this purpose, serotonin and tryptophan levels weredetermined, as well as the activity of key enzymes involved in theserotonin metabolism (tryptophan hydroxylase, tryptophan decarboxylase,indole amide dehydrogenase and monoamine oxidase) in the blood serum,brain and duodenum of animals which were fed a high-fat diet andinjected with Zn-64 stable isotope in aspartate form.

In general, the results of this study show a significant imbalance ofthe serotonergic system in obese animals occurring both in theperipheral and in central serotonergic systems, which can be one of thetrigger mechanisms for the development and progression of obesity.

In the peripheral system, serotonin is localized in enterochromaffincells of the gastrointestinal mucosa. It is there that about 80% to 95%of the total amount of this hormone in the body is synthesized.Serotonin is also synthesized in the pineal gland. Cote F. et al, PNASUSA.-2003.-Vol. 100.-P. 13525-13530. Eddahibi S. The serotonin pathwayin pulmonary hypertension./Eddahibi S., Adnot S.//Arch. Mai. Coeur.Vaiss.-2006.-Vol. 99-P. 621-625.

Serotonin synthesized in the intestine is stored in platelets; it isalso present in other peripheral tissues, such as the mammary gland,liver, bones, as well as in β-cells in the pancreas. Peripheralserotonin is involved in the regulation of intestinal movements,vasoconstriction processes and blood pressure. Serotonin also regulatesthe levels of glucose in the blood plasma, thrombogenesis, cardiacrhythm and the strength of heart contractions.

Significant inhibition of the peripheral serotoninergic system has beenestablished, evident in a decreased activity of all key enzymes againstthe background of depletion of the tryptophan pool. Despite theidentified changes, serotonin levels in the duodenum of animal models ofobesity significantly exceeded the values in the control group (Table11).

These results are generally consistent with the current concept that theintestinal serotonin correlates with the development of obesity and thatthe levels of serotonin in the intestines in obese people are increasedsignificantly. The accumulation of serotonin causes an increase in serumglucose concentrations, thus contributing to the development of diabetesand obesity.

TABLE 11 Main indicators of functioning of the peripheral serotonergicsystem (duodenum) in animals from experimental groups (M ± m, n = 10)Experimental groups Experimental variable C C + zinc DIO DIO + zincTryptophan, μg/g 169.2 ± 12.6 154.7 ± 13.6  76.3 ± 9.74 *  89.4 ± 8.99 *Serotonin, μg/g  6.56 ± 0.98  7.41 ± 0.54  14.6 ± 1.64 *  9.73 ± 0.82*,# Tryptophan hydroxylase activity,   398 ± 78   375 ± 61   210 ± 69 *  253 ± 58 * RU/mg protein Tryptophan decarboxylase activity,  0.81 ±0.25  0.79 ± 0.22  0.65 ± 0.23  0.78 ± 0.19 RU/mg protein Monoamineoxidase activity,  3.33 ± 0.04   3.82 ± 0.07  1.55 ± 0.07  1.72 ± 0.03RU/mg protein Indoleamine dioxygenase activity,  2348 ± 301  2150 ± 231  1378 ± 305 *  1768 ± 274 * μmol/mg protein * the difference issignificant versus the control group of animals; # the difference issignificant versus the group of animal models of obesity Note:C-control; C + zinc-control on the background of administration of Zn-64stable isotope in aspartate form; DIO-diet induced obesity; DIO +zinc-diet induced obesity on the background of administration of Zn-64stable isotope in aspartate form.

Analysis of the serum serotonin and tryptophan levels in animals havingobesity showed a significant decrease in the concentrations of bothsubstances, which may be a direct consequence of inhibition of thereactions of serotonin synthesis in enterochromaffin cells of thegastrointestinal mucosa (Table 12).

An additional factor contributing to low serotonin levels in the bloodis the activation of monoamine oxidase.

Modulation of the peripheral serotoninergic system can be a goodanti-obesity treatment strategy, as it can reduce obesity and increaseinsulin sensitivity.

Despite the importance of maintaining physiological levels of peripheralserotonin, it is central serotonin that plays a key role in theregulation of energy homeostasis. An inverse relationship betweencentral serotonin level and food intake was established. In the CNS,serotonin is synthesized in the hypothalamus and brainstem. It helpsregulate mood, sleep-wake cycles and diet.

Inhibiting serotonin synthesis in the brain via intraventricularinjection of p-chlorophenylalanine, an irreversible inhibitor oftryptophan hydroxylase, induces hyperphagia and weight gain in rats.Serotonin reuptake inhibitors and monoamine oxidase inhibitors reducefood intake. Thus, serotonin in the central nervous system functions asan anorexigenic neurotransmitter.

TABLE 12 Serum levels of tryptophan and serotonin and monoamine oxidaseactivity in animals from experimental groups (M ± m, n = 10)Experimental groups Experimental variable C C + zinc DIO DIO + zincTryptophan, μg/g 57.38 ± 5.73 56.12 ± 7.58 38.3 ± 6.72 * 66.65 ± 9.62Serotonin, μg/g  9.53 ± 0.72 10.16 ± 0.62 3.88 ± 0.34 *  6.33 ± 0.51 *,#Monoamine oxylase activity,  2.69 ± 0.06  2.52 ± 0.03 3.91 ± 0.04 * 3.11 ± 0.08 *,# RU/mg protein * the difference is significant versusthe control group of animals; # the difference is significant versus thegroup of animal models of obesity Note: C-control; C + zinc-control onthe background of administration of Zn-64 stable isotope in aspartateform; DIO-diet induced obesity; DIO + zinc-diet induced obesity on thebackground of administration of Zn-64 stable isotope in aspartate form.

The pathogenesis of obesity in rats was accompanied by a significantdrop in the tryptophan levels in the brain of animals, which may becaused by impaired transport of this amino acid across the blood-brainbarrier (Table 13).

It is known that the transport of aromatic amino acids andbranched-chain amino acids across the blood-brain barrier occurs withthe involvement of a specific carrier and is competitive, thereforeelevated serum concentrations of branched-chain amino acids, typical forobesity, will affect transport of tryptophan across the blood-brainbarrier. C. Newgard, J. An, J. Bain.//Cell Metabolism.-2009.-Vol. 9, No4.-P. 311-326. E. del Amo et al.,//european journal of pharmaceuticalsciences.-2008.-No 35.-P. 161-174.

TABLE 13 Main indicators of functioning of the central serotonergicsystem (brain) in animals from experimental groups (M ± m, n = 10)Experimental groups Experimental variable C C + zinc DIO DIO + zincTryptophan, μg/g 99.24 ± 8.53 87.71 ± 3.41  6.66 ± 1.08 * 31.56 ± 2.14*,# Serotonin, μg/g 27.26 ± 2.76 25.51 ± 2.71  7.4 ± 1.63 *  20.1 ± 3.21*,# Tryptophan hydroxylase   281 ± 27   291 ± 29   245 ± 18   286 ± 17activity, RU/mg protein Tryptophan decarboxylase  1.32 ± 0.43  1.63 ±0.41  2.19 ± 0.67  1.86 ± 0.37 activity, RU/mg protein Monoamine oxidaseactivity,  0.84 ± 0.08  0.91 ± 0.06   1.19 ± 0.07  0.99 ± 0.08 RU/mgprotein Indoleamine dioxygenase  1097 ± 285  1178 ± 267  2438 ± 290 * 2007 ± 175 * activity, μmol/mg protein * the difference is significantversus the control group of animals; # the difference is significantversus the group of animal models of obesity Note: C-control; C +zinc-control on the background of administration of Zn-64 stable isotopein aspartate form; DIO-diet induced obesity; DIO + zinc-diet inducedobesity on the background of administration of Zn-64 stable isotope inaspartate form.

Given a close metabolic connection between serotonin and tryptophan, anatural consequence of the lack of the latter will be a decrease inserotonin levels.

Impairment of serotoninergic transmission is one of the factorscontributing to the occurrence of depressive states and can beconsidered as one of the key causes of obesity. It is known that peoplewith congenital or acquired defects of the central serotonergic systemdevelop subjective negative reactions to starvation, which isaccompanied by a decrease in the production of serotonin. In such cases,even minor starvation may trigger the development of depressive states.Therefore, such people consume food in quantities that exceed theirphysiological needs.

Thus, low serotonin levels in the ventromedial and paraventricularnuclei of hypothalamus induce excessive food intake and cause insulinhypersecretion, which leads to a decrease in sensitivity of peripheraltissues to the action of this hormone and the development of insulinresistance.

Changes in the activity of enzymes involved in the serotonin metabolicpathway that were identified during this study contribute to furtherdepletion of serotonin reserves in the brain. Thus, a decrease in theactivity of tryptophanhydroxilase, an enzyme limiting the process ofserotonin synthesis, occurring on the background of activation ofmonoamine oxidase, an enzyme that ensures the degradation of serotonin,was observed.

Increased activity of indolamine-2,3-dioxygenase is indicative of theactivation of an alternative tryptophan metabolic pathway, which notonly contributes to further depletion of the pool of this amino acid,but also serves as a source of formation of a number of neurotoxiccompounds.

Administration of Zn-64 stable isotope in aspartate form to animals thatwere fed a high-fat diet led to normalization of most of theexperimental variables. Thus an increase in serotonin levels in thebrain due to an increase in tryptophan levels is observed and,accordingly, activation of serotonin synthesis against the background ofinhibition of an alternative way of its transformation and reduction inthe rate of serotonin degradation by monoamine oxidase. A similar effectwas found in the duodenum and serum.

Thus, the effect of zinc is complex and is implemented at the level offunctioning of both the central and peripheral serotonergic systems.These results substantiate the advisability of therapeutic use of zincpreparations as monotherapy or in combination with other drugs for theimprovement of overall metabolic status in the development of obesityand prevention of the obesity-related disorders.

Effects of Zn-64 Stable Isotope in Aspartate Form onProoxidant-Antioxidant Balance in Animal Models of Obesity

Free radical reactions that are necessary for the formation of enzymes,activation of transcription factors, oxidation of xenobiotics andbactericidal protection are the basis of normal functioning of the cell.In addition, they are involved in the expression of genes, transducehormonal and cellular signals and regulate the processes of cellreproduction. Thus, reactive oxygen species and reactive nitrogenspecies are produced naturally in the human body and are key by-productsin the metabolic process. Antioxidants maintain levels of free radicalswithin physiological limits. A balance between antioxidant defense andfree radical oxidation is necessary for proper function of cells. Whenthe amount of free radicals exceeds the activity of antioxidant defense,a phenomenon called oxidative stress is generated.

Oxidative stress and the resulting tissue damage and cell deathcontribute to many pathological conditions. Excess production of freeradicals and/or depletion of the defense systems leads to theprooxidant-antioxidant imbalance, which in turn causes damage to theprotein structures in cells, lipid bilayer of cell membranes and nucleicacids. Since the lipid bilayer is a component of all cell membranes,lipid peroxidation mediated by free radicals is one of the importantreasons for the cell membrane damage followed by the cell death.Degradation of membrane lipids causes an increased fluidity of the cellmembrane and its permeability to ions, disrupting cellular homeostasisas a whole. Products of free radical oxidation (4-hydroxyalkene, malonicdialdehyde, etc.) are highly mutagenic and cytotoxic.

Epidemiological, clinical and animal studies have shown that obesity isassociated with an altered redox state and an increased metabolic risk.In this case, oxidative stress may be not only a consequence, but also atrigger for the development of disorders in obese people. It isoxidative stress that is one of the factors that cause adipocytedysfunction. Excess oxidants, initially produced by a growing mass ofadipose tissue, stimulate sensitive to oxidative stress signalingpathways, which are mediated by the transcription factor NF-kB and JNKand p38-MAPK kinases, and activate a number of protein kinases (PKB,PKC, etc.).

In addition, oxidative stress activates preadipocyte differentiation andstimulates hypertrophy of mature adipose cells. Increased production ofROS in the accumulated adipose tissue further leads to the induction ofoxidative stress in the bloodstream, which contributes to the spread ofoxidative stress to organs distant from the fat depot.

Increased glucose levels, along with disorders of lipid metabolism andelevated concentrations of free fatty acid, determine the mechanisms offormation and progression of oxidative stress specific for obesity. Itis believed that hyperglycemia-induced oxidative stress occurs both as aresult of direct activation of ROS formation reactions and as a resultof disturbance of cell redox homeostasis.

The next phase of this study aimed to assess the prooxidant-antioxidantbalance in animal models of obesity treated with Zn-64 stable isotope inaspartate form.

The concentrations of lipid peroxidation products (LOPs) serve as aninformative criterion making it possible to draw a conclusion aboutintensity of oxidative processes. There are primary lipid peroxidationproducts (such as conjugated dienes) and secondary lipid peroxidationproducts (such as aldehydes, malonic aldehyde in particular), which areformed as a result of breakdown of carbon-carbon double bonds in thecarbon skeletons of oxidized molecules. Subsequently, the initiation oflipid peroxidation leads to the formation of conjugated Schiff bases ofphospholipids and malonaldehyde-like products, which cause disturbancesin the ordered orientation of phospholipid molecules and affectlipoprotein intermolecular interactions and configuration of thebasement membrane.

Considering the above, the concentrations of primary LOPs—conjugateddienes (CD), secondary products—TBA-reactive substances (TBARSs) and endproducts—Schiff bases (SB) were determined in animals treated with Zn-64stable isotope in aspartate form. Taking into account that obesity isaccompanied by the development of systemic oxidative stress which coversmost tissues to various extents and leads to the disruption of integrityof cell membranes and admission of lipid peroxidation products to thebloodstream, values characterizing the state of theprooxidant-antioxidant system were determined in the blood serum ofanimals and analyzed.

Elevated serum levels of primary products of free radical lipidoxidation (by 1.86 times) suggest that the initial phase of lipidperoxidation actively occurs even after ten weeks of experimentalobesity (Table 14). This result can be explained from the standpoint ofdisturbed lipid metabolism, namely impairment of the processes oftransportation of fatty acids and, accordingly, an increase in theplasma levels of free and esterified fatty acids, direct substrates forthe action of active oxygen species. On the other hand, accumulation oflipid peroxidation products in serum may be a direct result of violationof the integrity of cell membranes due to oxidative destruction of theirlipid component and permeability of lipid oxidation products into thebloodstream.

An additional factor that may contribute to increased oxidative stressin the development of obesity is a significant activation of MAO(demonstrated at the previous phase of this study), an enzyme involvedin the ROS production.

TABLE 14 Serum levels of lipid peroxidation products in animals fromexperimental groups (M ± m, n = 10) TBA-reactive substances, Conjugatednmol/mg protein dienes, Fe²⁺-ascorbate- Schiff bases, Experimentalnmol/mg Spontaneous induced RU/mg groups protein accumulationaccumulation protein C 0.021 ± 0.001 0.006 ± 0.0003 0.033 ± 0.005  41.31± 2.47 DIO 0.039 ± 0.002 * 0.029 ± 0.002 *  0.61 ± 0.003 * 168.86 ±8.15 * DIO + zinc 0.025 ± 0.008 0.005 ± 0.0003 #  0.15 ± 0.008 *,# 56.27 ± 4.33 *,# * the difference is significant versus the controlgroup of animals; # the difference is significant versus the group ofanimal models of obesity Note: C-control; C + zinc-control on thebackground of administration of Zn-64 stable isotope in aspartate form;DIO-diet induced obesity; DIO + zinc-diet induced obesity on thebackground of administration of Zn-64 stable isotope in aspartate form.

An increase in the levels of CD was accompanied by accumulation ofsecondary POLs, namely TBA-reactive substances. Thus, the serum levelsof TBA-reactive substances in animals having obesity were 4.8 timeshigher in comparison with the control value. In the case ofFe+2-ascorbate-dependent accumulation of TBA-reactive substances, thisvalue exceeded the result obtained in the group of control animals20-fold, which suggests a significant contribution of non-enzymaticreactions of initiation of lipid peroxidation processes toprooxidant-antioxidant imbalance in obese animals.

Such a significant increase in the concentrations of aldehyde POLs isregarded as an unfavorable marker, since these substances can bind toproteins to form stable adducts. Their formation may affect proteinfunction. In addition, proteins modified in this way have immunologicalproperties and may cause autoantibody production.

Changes in the levels of primary and secondary products were accompaniedby accumulation of the end products of lipid peroxidation, Schiff bases,which are formed as a result of condensation of aldehydes, includingmalondialdehyde, or ketones with amino groups of proteins and lead toimpairment of the structural and functional characteristics of thelatter. According to the obtained data (table), a significant increasein the levels of Schiff bases was observed in the serum of animalshaving obesity. Thus, this value was 4 times higher than in the group ofcontrol animals. The levels of the end products of lipid peroxidationcharacterizes the duration of oxidative homeostasis disorders,therefore, given a significant growth of this value in this study, thelong-term activation of free radical reactions may be discussed.

Thus, elevated levels of lipid peroxidation products on the 10th week ofobesity development clearly indicate that oxidative stress has asystemic nature and that this process is chronic, which is anunfavorable prognostic marker, as these metabolites are extremely toxiccompounds and their negative impact is exhibited at different levels andleads to DNA molecule damage, destruction of protein molecules andglycosaminoglycans, changes in the lipid composition of cell membranesand disruption of membrane-associated processes.

Activation of lipid peroxidation processes may indirectly indicate anincrease in the concentrations of ROS. An excess of ROS may directlyactivate a number of serine-threonine kinases, such as, PKC, AKT/PKB,mTOR, GSK-3 and p38 MAPK. These synergistically acting protein kinasesreduce insulin sensitivity of cells by selective phosphorylation ofserine and threonine residues in IRS molecules and contribute to thedevelopment of resistance of insulin-dependent cells to this hormone.

Administration of Zn-64 stable isotope in aspartate form to animalshelped normalize the levels of primary, secondary and end LOPs, whichserves as additional evidence of the ability of Zn-64 stable isotope inaspartate form to influence an overall prooxidant-antioxidant status ofthe body.

According to modern concepts, reactive oxygen species not only activatelipid peroxidation processes but also cause oxidative destruction ofprotein molecules, causing disruption of conformation of soluble andmembrane-bound enzymes, receptors and ion channels, which ultimatelyleads to the loss of their biological activity (enzymatic, receptor,transport, etc.). Oxidative modification of proteins and accumulation ofstructurally modified molecules is an important factor that maypotentially contribute to the production of new antibodies, thusprovoking an autoimmune reaction. Berlett B. S., Stadtman E. R. J. Biol.Chem.-1997.-V.272, No 33.-P.20313-20316.

Under the action of ROS, the native conformation of proteins isdisturbed resulting in the formation of large protein aggregates, orvice versa, fragmentation of protein molecules. Hydroxyl radicals mostoften cause protein aggregation and—in combination with superoxideanion—fragmentation with the formation of low molecular weightfragments. Lipid radicals may also cause fragmentation of proteinmolecules. The formation of carbonyl groups (aldehyde or ketone groupsof amino acid residues) may serve as a marker of oxidative proteindamage.

According to modern concepts, all amino acid residues in proteins may bemodified, but tryptophan, tyrosine, histidine and cysteine residues arethe most sensitive. ROS attack functional groups of amino acids thatmake up proteins, leading to the formation of primary amino acidradicals capable of interacting with neighboring amino acid residues. Ingeneral, a complex picture of the damaging effect of ROS on proteinmacromolecules is faced. Radicals, formed as a result of tyrosineoxidation, may interact with each other, forming bityrosine cross-linksin proteins. Bityrosine cross-links increase resistance of proteins tothe action of proteases, creating prerequisites for the accumulation offunctionally inactive proteins in the body. Oxidation of tryptophan isalso associated with the formation of covalent cross-links, which is anadditional factor that causes aggregation of protein molecules. Rojas V.C. et al., Arch. Med. Res.-1996.-V. 27, No 1.-P.1-6. Archakov A. I.,Mokhosoev I. M. Modification of proteins with active oxygen and theirdecomposition//Biochemistry.-1989.-Vol. 54, No 2.-P. 179-185.

Oxidative modification of proteins plays an important role in proteinmetabolism in the body. Accumulation of oxidized proteins is regarded asone of the factors regulating the synthesis and breakdown of proteinsand activation of multicatalytic proteases that selectively destroyoxidized proteins. A degree of oxidative damage to protein molecules maybe assessed by the accumulation of carbonyl derivatives, aldehyde- andketone-dinitrophenyl-hydrazones of a neutral nature in particular.Aldehyde-dinitrophenyl-hydrazones, detected at a wavelength of 356 nm,are early markers of oxidative degradation of proteins and indicate theinitial stages of damage to protein molecules under the action of freeradicals, while ketone-dinitrophenyl-hydrazones, which are detected at370 nm, are considered late markers of oxidative damage to proteins.

Since, unlike lipid peroxidation products, carbonyl derivatives are muchmore stable, this makes it possible to consider products of oxidativemodification of proteins as markers of oxidative damage in tissues.

Thus, an increase in the number of modified proteins may be consideredas an early criterion for the damage of tissues by free radicals and amarker of the depletion of antioxidant defense system in the body.

These studies revealed an increase in the serum levels of oxidativelymodified proteins in animal models of obesity (Table 15) with morepronounced changes in the levels of aldehyde-dinitrophenyl-hydrazonesthat indicate an active stage of the development of oxidative stress andmetabolic disorders accompanied by enhanced formation of free radicals.In general, elevated concentrations of carbonyl derivatives inoxidatively modified proteins in the serum of animals having obesityagainst the backdrop of intensification of lipid peroxidation processescan be regarded as indisputable evidence of prolonged oxidative stress.Thus, taking into account the data obtained, it can be said that thedevelopment of obesity is accompanied by activation of free radicaloxidation of proteins, which makes itself evident in the increasedamounts of carbonyl derivatives formed by oxidative modification ofproteins with absorption peaks at 356 and 370 nm.

TABLE 15 Serum levels of products of oxidative modification of proteinsin animals from experimental groups (M ± m, n = 10)Aldehyde-dinitrophenyl- ketone-dinitrophenyl- hydrazones, hydrazones,Groups nmol/mg protein nmol/mg protein C 0.187 ± 0.009 0.255 ± 0.023 DIO0.698 ± 0.041 * 0.571 ± 0.035 * DIO + zinc 0.253 ± 0.012 *,# 0.200 ±0.024 *,# * the difference is significant versus the control group ofanimals; # the difference is significant versus the group of animalmodels of obesity Note: C-control; C + zinc-control on the background ofadministration of Zn-64 stable isotope in aspartate form; DIO-dietinduced obesity; DIO + zinc-diet induced obesity on the background ofadministration of Zn-64 stable isotope in aspartate form.

The processes of modification of protein molecules by active oxygenspecies occur not only in pathological conditions. Thus, inphysiological conditions, there is a certain level of oxidativelymodified proteins in cells, reflecting the balance between the rate ofproteolytic degradation of these damaged, “used” molecules and the rateof their synthesis. In some cases, oxidative inactivation is a markerstage that increases sensitivity of proteins to the action of proteases,since proteolytic enzymes break down modified protein molecules muchfaster than native ones. Therefore, elevated levels of carbonylderivatives in animals having obesity may not only indicate thedevelopment of oxidative stress, but also give evidence of significantimpairments in the mechanisms of control and regulation of degradationof structurally modified proteins and, peculiarly, proteolytic enzymesthat ensure implementation of this process.

In animals that were fed a high-fat diet during the entire experimentand received injections of Zn-64 stable isotope in aspartate form, thelevels of aldehyde-dinitrophenyl-hydrazones exceeded the benchmark butwere lower compared to the values in untreated animals having obesity.As for keton-dinitrophenyl-hydrazones, their concentration remainedwithin the control value. Such results correlate with the data showing adecrease in the levels of LOPs and may suggest a decrease in theintensity of free radical oxidation reactions.

According to modern concepts, in cells, along with modification ofproteins by ROS, there is a so-called non-oxidative pathway for theformation of carbonyl derivatives, which consists in modifying proteinmolecules by aldehydes. Thus, the experiments of Burcham P. C. et al.showed that incubation of proteins with various aldehydes, includingMDA, induced an increase in the number of oxidatively modified proteinsin a concentration-dependent manner. Since administration of Zn-64stable isotope in aspartate form to obese animals resulted in a decreasein the levels of LOPs, it appears that the observed decrease in thedegree of oxidative modification of proteins may partly be associatedwith inhibition of the non-oxidative formation of carbonyl derivatives.

The basis of the positive effect of zinc on the overall oxidative statusthat was identified may be both its direct influence on the processes offree radical oxidation at the stage of initiation of chain reactions andits inclusion in the active centers of antioxidant enzymes. It is alsoimportant to mention a membrane stabilizing effect of this traceelement, which may be one of the mechanisms of its antioxidant action aswell. The role of zinc as an antioxidant is confirmed by its ability toact as an intramolecular stabilizer that prevents formation of disulfidestructures. In addition, zinc competitively replaces copper and ironions which trigger the formation of free radicals.

The free radical processes are controlled and regulated by antioxidantdefense (AOD), a complex multicomponent and multi-level system. Inphysiologically normal state, equilibrium between the levels of freeradical oxidation reactions and the activity of this system ismaintained, which ensures maintenance of lipid peroxidation processes ata stationary, rather low level.

The antioxidant defense system consists of non-enzymatic and enzymaticunits. Non-enzymatic antioxidants provide mainly rapid inactivation offree radicals of oxygen and nitrogen, while enzymatic antioxidants arereferred to the terminal system of long-term defense of the body.

Superoxide dismutase (EC1.15.1.1) is one of the key enzymes of AOD. Thisenzyme catalyzes a reaction of neutralization of superoxide anionradicals by their dismutation into less reactive molecules of hydrogenperoxide and triplet oxygen. SOD is the only among the most activeantioxidant enzymes that breaks the chains of oxygen-dependentfree-radical reactions in the cells of aerobic. Poberezkina N. B.,Osinskaya L. F. Biological role of superoxide dismutase//Ukr. biohimjournal.-1989.-Vol. 61, No 2.-P. 14-27. Dudochnik L. B., Tikhaze A. K.,Alesenko A. V. et al. Change in the activity of superoxide dismutase andglutathione peroxidase in the process of lipid peroxidationintensification in liver ischemia//Bul. exp. biol. Med.-1981.-Vol.XCI,No 4.-P. 451-453.

Given the leading role of SOD in reactive oxygen species metabolism anda significant contribution of superoxide anion radicals to the inductionand development of oxidative stress, the activity of Cu—Zn-dependent SODin the serum of animal models of obesity treated and untreated withZn-64 stable isotope in aspartate form was investigated.

In this experiment, a statistically significant decrease in SOD activitywas found in animals that ate a high-fat diet. Administration of Zn-64stable isotope in aspartate form to experimental animals caused anincrease in SOD activity not only in comparison with the values inuntreated animal models of obesity, but also relative to the control(Table 16).

TABLE 16 Serum superoxide dismutase and catalase activities in animalsfrom experimental groups (M ± m, n = 10) Experimental groups C DIO DIO +zinc Superoxide dismutase activity, 3.36 ± 0.36 2.65 ± 0.41 *  4.5 ±0.43 *,# RU/min per mg protein Catalase activity, 0.52 ± 0.05 0.43 ±0.02* 0.48 ± 0.02# umol H₂O₂/min per mg protein * the difference issignificant versus the control group of animals; # the difference issignificant versus the group of animal models of obesity Note:C-control; C + zinc-control on the background of administration of Zn-64stable isotope in aspartate form; DIO-diet induced obesity; DIO +zinc-diet induced obesity on the background of administration of Zn-64stable isotope in aspartate form.

Taking into account a certain zinc deficiency characteristic of thepathogenesis of obesity, restoration of the activity of this enzymefollowing the administration of the solution of Zn-64 stable isotope inaspartate form may be a consequence of normalization of its levels inthe body and its active involvement in the regulation and synthesis ofzinc-dependent enzymes, SOD in particular.

A decrease in SOD activity can be viewed as a consequence of a certaindepletion of the antioxidant defense system due to gradual damage of itscomponents by free radicals and LOPs. Thus, according to modernconcepts, activity of this enzyme is closely related to the intensity ofLOP processes since excessive accumulation of toxic secondary productsof lipid oxidation causes inhibition of the activity of SOD and otherenzymes of the antioxidant system. Literature review and data analysison the involvement of ROS in the oxidative degradation of proteinssuggest that a decrease in the enzymatic activity of SOD may be due toan oxidative modification of the enzyme molecule. Since SOD is ametal-containing enzyme, protein-damaging oxygen radicals can be formeddirectly in the active center of the enzyme. In this case, the hydroxylradical, OH⁻, formed in the reactions of Fenton and Haber-Weiss fromhydrogen and superoxide, acts as a direct agent that inactivates theenzyme. Thus, the experiments of Salo D.C. et al. show that incubationof superoxide dismutase in a medium containing oxygen radicals leads tothe cleavage of an enzyme molecule resulting in the formation ofadditional protein fractions. The authors explain the result byoxidative inactivation of superoxide dismutase when exposed to H202.Such a view is consistent with the ability of copper atoms in the activecenter of the enzyme to accelerate formation of free radicals. Anotherargument in favor of the involvement of metals with several commonoxidation states in SOD inactivation is the fact that Mn2+-containingsuperoxide dismutase is not susceptible to oxidative degradation whenincubated with H₂O₂.

Since an important antioxidant enzyme in the system is catalase, whichneutralizes hydrogen peroxide formed as a result of dismutation of thesuperoxide anion radical by superoxide dismutase, the activity of thisenzyme in animal models of obesity treated and untreated with Zn-64stable isotope in aspartate form was assessed. Similar to a decrease inSOD activity, an inhibition of catalase activity in untreated obeseanimals was observed.

A decrease in the activity of both enzymes against the background ofactivation of lipid peroxidation processes is regarded as a negativeprognostic event contributing to further escalation of free radicalprocesses.

Administration of Zn-64 stable isotope in aspartate form caused a slightincrease in the activity of catalase when compared with the resultsobtained from the group of untreated animal models of obesity, whichgenerally correlated with the previously established normalization ofprooxidant-antioxidant balance.

Normalization of the activities of the studied antioxidant enzymesfollowing administration of the test substance is consistent with theliterature data on other forms of this ion. Thus, it was shown that thepreliminary administration of Zn-64 stable isotope in aspartate form ledto an increase in the activity of SOD and catalase in rat hepatocytes inethanol intoxication.

A positive effect of Zn-64 stable isotope in aspartate form on theactivity of key antioxidant enzymes that was revealed may be explained,first of all, by an increase in their synthesis due to an increase inthe concentrations of zinc, a necessary structural element ensuring aproper functional activity of these enzymes.

The results obtained regarding the normalizing effect of Zn-64 stableisotope in aspartate form on the prooxidant-antioxidant balance confirma significant antioxidant potential of this isotope. Although zinc doesnot belong to typical antioxidants that can directly interrupt freeradical reactions, it has an indirect effect on the overallprooxidant-antioxidant balance. Thus, zinc is an inhibitor of NADPHoxidases, a group of enzymes involved in the formation of the aggressivesuperoxide anion radical.

Directly interacting with the sulfhydryl groups of proteins, zincprotects them from oxidation by active oxygen species, induces thesynthesis of metallothioneins, cysteine-rich metal-binding proteins thatact as a trap for radicals, helps to inhibit the formation of reactivemixed valence metal oxides and exhibits a membrane stabilizing effect.It has been found that dietary zinc deficiency decreases concentrationsof vitamin E in plasma and some other organs, which affects the generalantioxidant reserve of the body.

Another mechanism for the implementation of antioxidant properties ofZn-64 stable isotope in aspartate form is associated with its ability tostabilize cell membranes, which is especially important in progressiveoxidative stress. A possible cause of the membrane-protective effect ofzinc may be explained by its mediating role in the induction ofsynthesis of metallothioneins directly involved in the detoxification ofheavy metals and stabilization of membranes. Elevated levels ofmetallothioneins may maintain the integrity of membranes and protectcells from the action of alkylating agents.

It has been found that zinc can stabilize the cell membrane byinfluencing the synthesis of phospholipids (activation of PS synthase,PS decarboxylase, PEA methyltransferase and phospholipidmethyltransferase with a simultaneous decrease in the activity of PHIsynthetase) and their asymmetric distribution.

Thus, given the importance of controlling intensity of free radicalreactions and maintaining a proper antioxidant status of the body,especially for patients with systemic chronic diseases pathogenesis ofwhich is closely associated with the development of oxidative stress,the use of Zn-64 stable isotope in aspartate form as an additional meansin the basic therapy of the disease may contribute to the improvement ofan overall metabolic status.

Effects of Zn-64 Stable Isotope in Aspartate Form on Cytokine Profile,Resistin and Ghrelin Levels in Animal Models of Obesity

There is no doubt that adipose tissue is not only an energy depot of thebody, but also an organ that is actively involved in the regulation ofmetabolism through a complex of endocrine, paracrine and autocrinesignals modulating responses of many tissues and organs, including thehypothalamus, hypophysis, pancreas, liver, skeletal muscles, kidneys,endothelium, the immune system, etc. Thus, adipose tissue secretes morethan 50 protein factors, hormones and growth factors, includingcytokines. There are pro-inflammatory cytokines, such as IL-1, IL-6,IL-8, IL-12, TNF-α, IFN-γ and anti-inflammatory cytokines, such as IL-4,IL-10, IL-13, TGF Mohamed-Ali V., Pinkney J., Coppacf S. Adipose tissueas endocrine and paracrine organ//Int J Obes Relat Meabol Disord 1998;22: 1145-1158.

One of the consequences of excessive production of reactive oxygenspecies in adipocytes is the initiation of signaling cascades, leadingto an increase in the production of pro-inflammatory cytokines bymacrophages which infiltrate in adipose tissue increasing in its mass.The result of such disorders is the formation of systemic chronicinflammation in the body of a person that develops obesity. According tothe actively discussed modern concept, it is subclinical chronicinflammation in adipose tissue that is thought to be one of the keylinks in the pathogenesis of obesity and obesity-related diseases.Chronic inflammation of adipose tissue is characterized by cellularinfiltration, fibrosis, microcirculation changes, impaired adipokinesecretion and adipose tissue metabolism disorders, as well as increasedblood levels of such non-specific inflammatory markers as C-reactiveprotein, fibrinogen, and leukocytes Raj ala M., SchererE.//Endocrinology 2003; 144: 3765-3773.

An increase in the levels of pro-inflammatory cytokines not only inadipose tissue, but also in blood serum occurs a result of theinflammatory process in adipose tissue.

Cytokines, as endogenous biologically active mediators that regulateintercellular and intersystem interactions, have an effect on thesurvival of cells by regulating their growth, differentiation,functional activity, and apoptosis. They ensure coordination of actionsof the immune, endocrine and nervous systems under physiologicalconditions and in response to pathological effects. It was previouslybelieved that cytokines were produced by lymphocytes, monocytes andtissue macrophages. However, the results from recent research show that,in obesity, as in any inflammatory process, infiltration of neutrophils,T-lymphocytes, and then resident macrophages into adipose tissue occursat an early stage, which determines the initial mechanisms ofinflammation. It has been shown that macrophages contribute tohypertrophy of adipocytes, which is accompanied by an increase in theirfunctional activity and increased synthesis of cytokines and leads tofurther intensification of the inflammatory response. Hypertrophiedadipocytes intensely secrete chemokines and their receptors, whichstimulate the influx of new neutrophils, macrophages and lymphocytes,thus contributing to a further increase in adipocyte hypertrophy,preservation and intensification of the inflammatory response.Adipocytes increase the secretion of cytokines by macrophages, which inturn act on adipocytes, causing hypertrophy and activation of adiposetissue cells. It has been found that hypertrophied adipocytes, likelymphocytes and macrophages, produce cytokines and activate thecomplement, triggering a chain of inflammatory processes. As a result,the inflammation becomes steady and systemic. In addition, lipidperoxidation products, such as trans-4-oxy-2-nonenal and malonicdialdehyde, are chemoattractants for monocytes and macrophages.Strengthening of the processes of lipid peroxidation in accumulatedadipose tissue contributes to the attraction and infiltration ofmacrophages into adipose tissue in obesity, thus actively contributingto the launch of inflammation reactions.

Consequently, an increasing adipose tissue mass is a constant source ofpro-inflammatory cytokines synthesized both by adipocytes andmacrophages incorporated into adipose tissue, which leads to theformation of a chronic inflammatory process and maintenance ofinflammation in the body. Its low intensity does not give directclinical symptoms, but at the same time, this process is systemic innature, which means that it affects a wide range of organs and tissuescausing changes in their metabolism and impairing their function andimmune system reactions.

Given the above, the next phase of the study was to find out whether theadministration of Zn-64 stable isotope in aspartate form has an effecton the cytokine profile in obese animals. For this purpose,concentrations of the main pro-inflammatory (IL-1, IL-6, IL-12, IFN-γ)and anti-inflammatory (IL-4, IL-10, TGF) cytokines in adipose tissue andserum of experimental animals were determined, which allowed us to makea conclusion about the intensity of the inflammatory process in adiposetissue and assess whether such inflammatory process is systemic.

According to the obtained results, the development of obesity wasaccompanied by an increase in the levels of all analyzedpro-inflammatory cytokines (Table 17) in the adipose tissue of animalsfed a high-fat diet, which indicates activation of the inflammatoryprocess.

In turn, a prolonged inflammatory process may lead to the development ofvarious complications and be a risk factor for insulin resistance anddiabetes. Cytokines are not only able to reduce sensitivity of cells toinsulin action, but also intensify inflammatory processes and increaseaccumulation of inflammatory intermediates, causing tissue damage andorgan dysfunction J. Hirosumi et al.//Nature.-2002.-Vol. 420, No6913.-P. 333-336. C. Jiang, W. Wang, J. Tang.//Journal ofEndocrinological Investigation.-2013.-Vol. 36, No 11.-P. 986-992.

TABLE 17 Cytokine profile in the adipose tissue of animals fromexperimental groups (M ± m, n = 10) Groups Levels, RU/mg proteinPro-inflammatory cytokines Anti-inflammatory cytokines IL-1 IL-6 IL-12IFN-γ IL-4 IL-10 TGF C 5.6 ± 1.3 5.9 ± 0.7  1.2 ± 0.03 4.7 ± 1.2 4.8 ±0.5 4.7 ± 0.7 4.5 ± 0.9 C + zinc 4.9 ± 1.7 5.4 ± 0.3  1.0 ± 0.03 5.0 ±0.8 5.0 ± 0.4 4.9 ± 0.2 5.1 ± 0.6 DIO 9.8 ± 2.8* 8.9 ± 0.7* 2.87 ± 0.08*7.6 ± 1.2* 3.9 ± 0.2* 3.1 ± 0.6* 3.1 ± 0.1* DIO + 5.8 ± 1.8# 6.1 ± 0.7#1.99 ± 0.01# 4.9 ± 0.8# 5.1 ± 1.2# 5.8 ± 0.8# 5.2 ± 0.9# zinc *thedifference is significant versus the control group of animals; #thedifference is significant versus the group of animal models of obesityNote: C—control; C + zinc—control on the background of administration ofZn-64 stable isotope in aspartate form; DIO—diet induced obesity; DIO +zinc—diet induced obesity on the background of administration of Zn-64stable isotope in aspartate form.

It has been proven that high levels of pro-inflammatory cytokines,including those mentioned above, can provoke apoptosis of β-cells. Highconcentrations of IL-12, the expression of which is activated by IFN-γ,lead to infiltration of CD8+lymphocytes in the pancreas and thedevelopment of acute pancreatitis. IL-1β, via binding to specificreceptors on the surface of these cells, causes activation ofNF-κB-mediated apoptosis, which leads to DNA fragmentation and loss offunctional activity of cells. In addition, IL-1β may also be regarded asone of the factors contributing to the development of resistance ofperipheral tissues to insulin. IL-1β has been shown to activate IκBkinase-β which has an effect on insulin signaling by phosphorylating aserine residue in the insulin receptor substrate (IRS)-1. In addition,IL-10 is able to increase resistance to the action of insulinindirectly, by activating lipogenesis in the liver and contributing toan increase in the levels of triglycerides and free fatty acids inadipocytes.

It has been shown that IL-6 is accumulated in direct proportion to anincrease in the adipose tissue mass in peripheral blood. Adipocytes arethe second largest source of IL-6 after the immune system: 35% ofcirculating IL-6 is synthesized by adipose cells. Its concentration inthe blood is directly proportional to the body mass index and isincreased in obesity. At the same time, a decrease in body weight isaccompanied by a decrease in the blood levels of IL-6. When in excess,IL-6 exacerbates insulin resistance by suppressing synthesis of one ofthe insulin receptor subunits. By activating lipolysis in visceraladipose tissue, IL-6 contributes to the progressive development of fattyhepatosis and systemic atherosclerosis. In addition, IL-6 inducesincreased production of C-reactive protein (CRP), another factorassociated with obesity V. Rotter et al.//the Journal of BiologicalChemistry.-2003.-No 278.-P. 45777-45784. Fantuzzi G./Journal of Allergyand Clinical Immunology.-2005.-Vol. 115, No 5.-P. 911-919.

One of the controlling mechanisms for the levels and, accordingly, thebiological effects of pro-inflammatory cytokines, is implemented by agroup of anti-inflammatory cytokines. These cytokines are able toinhibit the synthesis of pro-inflammatory cytokines by affectingtranscription of specific genes, induce the synthesis of receptorantagonists of interleukins RAIL, enhance the production of solublereceptors and reduce the density of pro-inflammatory receptors on cells.Therefore, to clarify possible mechanisms of the effects of Zn-64 stableisotope in aspartate form on the profile of pro-inflammatory cytokines,the levels of IL-4, IL-10, and TGF were determined.

Detected changes in the levels of pro-inflammatory cytokines occurredagainst the background of a slight decrease in the levels ofanti-inflammatory cytokines in obese animals. At the same time, inanimals treated with Zn-64 stable isotope in aspartate form, the levelsof anti-inflammatory cytokines were not only higher than in theuntreated animal models of obesity, but also higher than in the animalsfrom the control group.

It should be emphasized that the absence of changes in the animals fromthe control group treated with the test substance suggests that along-time use of Zn-64 stable isotope in aspartate form is safe and itis able to show a therapeutic effect only with the development ofpathological conditions.

As mentioned above, the pathogenesis of obesity is accompanied by asystemic chronic inflammatory process, the intensity of which can beassessed by the serum levels of pro- and anti-inflammatory cytokines.

Analysis of the cytokine profile in the serum of animals having obesity(Table 18) showed an increase in the levels of pro-inflammatorycytokines, more pronounced compared with the data obtained from adiposetissue. No statistically significant changes in the levels ofanti-inflammatory cytokine IL-4 were found. A slight increase in theserum levels of IL-10 in obese animals can be regarded as a certaincompensatory response of the body to a metabolic disorder.

In animals treated with Zn-64 stable isotope in aspartate form, therewas a decrease in the levels of pro-inflammatory cytokines against thebackground of an increase in the levels of anti-inflammatory cytokines,which were even higher than in the animals from the control group.

TABLE 18 Cytokine profile in the serum of animals from experimentalgroups (M ± m, n = 10) Groups Levels, RU/mg protein Pro-inflammatorycytokines Pro-inflammatory cytokines IL-1 IL-6 IL-12 IFN-γ IL-4 IL-10TGF C  3.4 ± 0.3 4.5 ± 0.3 0.5 ± 0.05 3.6 ± 0.8 5.1 ± 0.2 3.9 ± 0.4 3.8± 0.8 C + zinc  3.5 ± 0.7 4.3 ± 0.2 0.3 ± 0.04 4.6 ± 0.6 4.6 ± 0.8 4.1 ±0.5 4.1 ± 0.4 DIO 11.1 ± 2.0* 7.9 ± 0.5* 3.7 ± 0.07* 6.5 ± 0.8* 4.4 ±0.9 4.1 ± 1.5 3.5 ± 1.3 DIO +  4.2 ± 0.4# 5.1 ± 0.4# 2.4 ± 0.06*, # 4.1± 1.2 5.6 ± 1.6 6.8 ± 1.1*, # 5.7 ± 0.3*, # Zinc *the difference issignificant versus the control group of animals; #the difference issignificant versus the group of animal models of obesity Note:C—control; C + zinc—control on the background of administration of Zn-64stable isotope in aspartate form; DIO—diet induced obesity; DIO +zinc—diet induced obesity on the background of administration of Zn-64stable isotope in aspartate form.

One of the basic mechanisms of the effect of zinc on the cytokineprofile may be its inhibition of transcription factors sensitive tooxidative stress. Zinc may also partially block genes encodingpro-inflammatory cytokines, such as IL-6 and IL-8.

A certain normalizing effect of Zn-64 stable isotope in aspartate formon the cytokine profile in animal models of obesity may serve asevidence of the possible anti-inflammatory potential of the studied testsubstance in obesity.

Thus, a decrease in the levels of pro-inflammatory cytokines in theserum and adipose tissue of animals treated with Zn-64 stable isotope inaspartate form may in part be due to an increase in the levels ofanti-inflammatory cytokines. Taking into account the existence of aclose relationship between the amount of adipose tissue and the levelsof pro-inflammatory cytokines that it produces, it appears that that therevealed positive effect of Zn-64 stable isotope in aspartate form onthe cytokine profile is associated primarily with its influence on bodyweight, and therefore on the amount adipose tissue.

In addition to cytokines, adipocytes secrete a number of biologicallyactive substances involved in the regulation of energy metabolism. Oneof such substances is resistin or adipocyte-specific secretory factor(ADSF/FIZZ3). Participation of resistin in stimulation of the mechanismsof inflammation, activation of the endothelium and proliferation ofvascular smooth muscle cells makes it possible to view it as a marker oreven an etiological factor in the development of diseases. Thisadipose-derived hormone has a feedback effect on the fat metabolism: onthe one hand, its concentrations increase with the differentiation ofadipocytes, and on the other hand, resistin suppresses adipogenesis.Resistin, as one of the causes of insulin resistance, can be a linkbetween obesity and the development of diabetes. According to theliterature data, resistin levels may be used as a predictor ofsusceptibility to type II diabetes and obesity. It has been shown thatresistin is capable of reducing sensitivity of peripheral tissues to theaction of insulin, thus stimulating the development of insulinresistance. Resistin activates NF-κB-dependent expression and release ofpro-inflammatory cytokines and adhesion molecules, including TNF-α andIL-6 Fantuzzi G./Journal of Allergy and Clinical Immunology.-2005.-Vol.115, No 5.-P. 911-919. C. Jiang et al.,//Journal of EndocrinologicalInvestigation.-2013.-Vol. 36, No 11.-P. 986-992.

Therefore, the levels of resistin in the adipose tissue and serum ofanimal models of obesity from all experimental groups were investigated.In accordance with the results obtained in the experiment, there was atendency to an increase in the levels of this adipokine, more pronouncedin adipose tissue (Table 19).

TABLE 19 Resistin and ghrelin levels in adipose tissue and serum ofanimals from experimental groups (M ± m, n = 10) Resistin, Ghrelin,Experimental RU/mg protein RU/mg protein groups Adipose tissue SerumSerum C 0.26 ± 0.05 0.18 ± 0.05 0.019 ± 0.003 C + zinc 0.25 ± 0.05 0.12± 0.05 0.016 ± 0.003 DIO 0.34 ± 0.05* 0.19 ± 0.05 0.027 ± 0.003* DIO +zinc 0.25 ± 0.05# 0.16 ± 0.05 0.019 ± 0.003# *the difference issignificant versus the control group of animals; #the difference issignificant versus the group of animal models of obesity Note:C-control; C + zinc-control on the background of administration of Zn-64stable isotope in aspartate form; DIO-diet induced obesity; DIO +zinc-diet induced obesity on the background of administration of Zn-64stable isotope in aspartate form.

Taking into account that resistin is a promoter of the maturation of fatcells and acts as an autocrine regulator of the formation of adiabaticfactors in adipose tissue, even a slight increase in the levels of thisadipokine will contribute to the growth of adipose tissue andprogression of obesity-associated metabolic disorders.

The levels of resistin in animal models of obesity injected with Zn-64stable isotope in aspartate form were within the control values.

Since resistin is mainly secreted by preadipocytes and, to a lesserextent, by mature adipocytes of visceral adipose tissue, the positiveeffect of Zn-64 stable isotope in aspartate form can be explained by itsability to have an effect on the body weight of animals, and hence theaccumulation of fat mass in animals maintained on a high-fat diet.

Another factor directly involved in the regulation of appetite isghrelin, a lipophilic hormone secreted mainly by P/D1 cells lining thefundus of the stomach, and, to a lesser extent, by other organs, such asthe hypothalamus, hypophysis, gonads and ε-cells of the islets ofLangerhans. This peptide plays an important role in the regulation ofhunger and energy metabolism, stimulating food intake and provoking thedevelopment of obesity. Ghrelin receptors are localized in the samehypothalamic structures as the leptin receptor, Ob-Rb, and in thearcuate and ventromedial nuclei. Its high levels may contribute tolong-term weight gain. When the threshold of ghrelin level in the bodyis lowered, appetite decreases F. Ferrini et al.,//CurrentNeuropharmacology.-2009.-Vol. 7, No 1.-P. 37-49. J. Camiña etal.,//Endocrine.-2003.-Vol. 22, No 1.-P. 5-12.

Studies conducted over the past few years have revealed the importanceof ghrelin in the regulation of energy balance in the body through theinfluence of this hormone on the hypothalamus with the involvement ofneuropeptide Y and the endocanabinoid system.

Ghrelin expression is enhanced in response to hypoglycemia and isinhibited during hyperglycemia. This may indicate that ghrelin combinesthe body's metabolic and hormonal responses to fasting, with theinvolvement of insulin and the mechanisms that maintain a proper serumglucose concentration. Ghrelin has orexigenic, adipogenic andsomatotropic properties and acts as a leptin antagonist, increasing theneed for food. It has been shown that active immunization againstghrelin causes weight loss. The orexigenic effect of ghrelin consists inits ability to increase neuropeptide-Y neuronal activity and inhibitproopiomelanocortin neurons.

Ghrelin receptors are located both in the central nervous system(hypophysis, hypothalamus) and in other organs (pancreas, intestine,stomach). This peptide plays an important role in the regulation ofhunger and energy metabolism, stimulating food intake and provoking thedevelopment of obesity. Its levels increase in fasting, weight loss,calorific food intake and hypoglycemia. Elevated plasma levels ofghrelin after weight loss caused by diet are consistent with thehypothesis that ghrelin plays a role in the long-term regulation of bodyweight. The levels of ghrelin are reduced in people with obesity, type 2diabetes and hypertension.

Given a close relationship between the impaired energy homeostasis andthe development of obesity, the serum levels of ghrelin in animal modelsof obesity treated and untreated with Zn-64 stable isotope in aspartateform were analyzed.

The data show (FIG. 16) that the development of obesity was accompaniedby a decrease in the levels of this hormone, and the administration ofZn-64 stable isotope in aspartate form to animals in the control groupalso caused a decrease in its serum levels. In general, such data areconsistent with information provided in the literature, because it isknown that the levels of circulating ghrelin inversely correlate with apositive energy balance, total body weight and adipose tissue mass,adipocyte size and leptin levels. For example, the levels of ghrelin inpatients with anorexia are higher than in patients that develop obesity.In animal models of obesity injected with the test substance, the levelsof ghrelin were similar to those determined in animals from the controlgroup M. Kojima, K. Kangawa.//Physiological Reviews.-2005-Vol. 85, No2.-P. 495-522.

Thus, the data suggest that Zn-64 stable isotope in aspartate form has anormalizing effect on the functional status of adipocytes, which mayresult in the restoration of cytokine balance to the physiologicallynormal state.

Levels of Divalent Metal Ions in the Organs of Animal Models of Obesity

Participation of zinc in physiological and pathophysiological processesdepends largely on its levels in the body. Zinc is found in all cellsand organs, but its concentrations in one or another organ varyconsiderably and depend on the specific activity of the organ. Zincreserves in the human body are quite small and amount to about 1.5 to 3g. This figure depends on many factors: the age and sex of a person, thecondition of gastrointestinal mucosa, associated diseases, pregnancy,etc. Zinc is found in almost all tissues. About 62-63% of zinc reservesare in skeletal muscles. According to the data provided by a number ofresearchers, zinc is distributed in the human body as follows (μg/g):skin, adrenal glands—6, ovaries—12, brain—13, lymph nodes—14, GItract—21, heart—27, kidneys—37, liver—38, muscles—48, bones—66, prostategland—87, sperm—125. Whole blood contains about 2.5 to 5.3 μg/ml ofzinc. There is less zinc in plasma (0.7 to 1.2 μg/ml, which is about 0.2to 1% of the total amount of zinc in the body). The levels of zinc inblood serum are slightly higher (1.1 to 1.3 μg/ml) than in plasma due tothe destruction of red blood cells.

The levels of zinc and some divalent metals (copper and manganese) weredetermined in skeletal muscles, kidneys and liver of animals havingobesity, as well as the effects of Zn-64 stable isotope in aspartateform on the levels of these metals.

The choice of copper and manganese for analysis is explained by theirexceptional importance and involvement in key metabolic processes. Inaddition, the studied metals have an effect on the digestibility andbioavailability of each other, so an abnormal level of one of them isoften the result or cause of changes in the levels of the other metalsmentioned above. Most often they compete with each other. Thus, forexample, iron present in food in large amounts reduces the absorption ofzinc by about 2-fold. The presence of copper also reduces the absorptionof zinc in the gastrointestinal tract due to its competitive associationwith transport metalloenzymes.

TABLE 20 Levels of divalent metal ions in the muscles, kidneys and liverof animals from experimental groups (M ± m, n = 10) Levels, μg/g tissueZinc Manganese Copper Muscles C  7.63 ± 0.66 — 0.72 ± 0.05 DIO  6.82 ±0.63 — 1.06 ± 0.12* DIO + zinc  9.10 ± 0.42*,# — 0.72 ± 0.15# Kidneys C18.97 ± 0.52 0.75 ± 0.02 4.74 ± 0.49 DIO 21.73 ± 1.02* 0.63 ± 0.05* 4.41± 0.51 DIO + zinc 20.28 ± 1.75 0.64 ± 0.03* 4.94 ± 0.82 Liver C 27.64 ±1.47 2.21 ± 0.24 3.99 ± 0.21 DIO 21.75 ± 1.12* 1.58 ± 0.12* 2.56 ± 0.11*DIO + zinc 31.43 ± 1.78*,# 1.94 ± 0.12# 3.93 ± 0.09# *the difference issignificant versus the control group of animals; #the difference issignificant versus the group of animal models of obesity Note:C-control; C + zinc-control on the background of administration of Zn-64stable isotope in aspartate form; DIO-diet induced obesity; DIO + zinc-diet induced obesity on the background of administration of Zn-64 stableisotope in aspartate form.

Copper belongs among the essential trace elements. The body of an adultcontains about 110-150 mg of copper. Half of this amount is in themuscles. Smaller reserves of copper are concentrated in the liver, thegray matter in the brain hemispheres and in the bone marrow. Thisimportant trace element is found in enzymes and proteins (mainlyceruloplasmin). The latter is a metalloenzyme that catalyzes oxidationof a number of biologically active substances and ensures the deliveryof copper to various tissues and organs. It is known that copper is aspecific activator of cytochrome oxidase, tyrosinase and copper oxidase.Copper activates arginase and aminopeptidase, enzymes of proteinmetabolism, which enhance the synthesis of nucleic acids necessary forhealing processes. This biotic is involved in the processes of bloodformation, hemoglobin synthesis, function of the cytochrome system andis part of red blood cell stroma. Copper used in microdoses increasesglycogen levels in skeletal muscles and liver. It shows an insulin-likeactivity, accelerating the glucose oxidation and inhibiting thebreakdown of glycogen. Copper strengthens the neutralizing function ofthe liver and normalizes mineral metabolism. Copper is part of manyenzymes, it determines their function and regulates their action. It ispart of all oxidases, and as such is an important element of redoxreactions in the body. These enzymes are necessary for the processes ofcellular respiration and protection of cells from the effects of freeradicals and are involved in the synthesis of myelin, biosynthesis ofconnective tissue, and metabolism of glands. The antioxidant activity ofcopper is associated with its participation in the structural formationof superoxide dismutase.

Manganese is an essential trace element necessary to ensure a propermetabolic status of the body. Its highest levels are found in the bones,liver and gray matter in the brain hemispheres. This biotic has aninsulin-like effect, reducing the blood glucose levels and increasingthe synthesis of glycogen. It has been found that manganese stimulatesblood formation. It has a high oxidative activity and a pronouncedlipotropic (cholin-like) action. This trace element has an effect on thefat and protein metabolism and the synthesis of a number of vitamins. Inaddition, it is part of the most important enzymatic systems. Manganesesalts weaken a hypertensive effect of adrenaline and induce a decreasein adrenaline hyperglycemia. Manganese has cholesterol-lowering andanti-sclerotic effects.

According to the results obtained (Table 20), there was a tendencytowards redistribution of zinc between the organs in the development ofobesity. Thus, the zinc levels increased slightly in the kidneys anddecreased in the liver and muscles. An increase in the zinc levels inthe muscles of animals treated with Zn-64 stable isotope in aspartateform is, on the one hand, quite natural, given that most of all zincreserves in a normal physiological condition are located in skeletalmuscles, and on the other hand, this can be considered as indirectconfirmation of the absence of visible functional impairments in theprocesses of transportation and deposition of zinc in obese animals thatwere administered the test substance.

More significant is a decrease in the zinc levels in the liver of animalmodels of obesity. After all, the liver is the main place for thesynthesis of zinc-containing proteins. Therefore, a decrease in zincreserves in this organ will contribute to the development andprogression of disorders associated with insufficiency of zinc-dependentand zinc-containing enzymes. One of the consequences of reducing zinclevels in the liver can be a decrease in the serum activity ofsuperoxide dismutase in obese animals that was identified at theprevious phase of this study.

Zinc levels in the liver of animals fed a high-fat diet and injectedwith Zn-64 stable isotope in aspartate form were even slightly higherthan in animals from the control group, which is indicative of therestoration of zinc homeostasis.

As for copper, there was a tendency towards an increase in its levels inthe muscles and a decrease in the kidneys. The most pronounced changesin the copper concentrations were observed in the liver, an organ thatplays a major role in the metabolism of this trace element.

It should be emphasized that zinc is a competitor of copper in theprocesses of absorption in the intestines and when at highconcentrations, it may cause development of copper deficiency in thebody. Therefore, an absence of significant changes in the copper levelsin animals treated with Zn-64 stable isotope in aspartate form indicatesthat the dose level of this element was selected properly.

These results suggest a positive effect of Zn-64 stable isotope inaspartate form on key systems directly involved in the development andprogression of obesity.

In general, summarizing the results, Zn-64 stable isotope in aspartateform administered to animal models of obesity exhibits a complex effectthat extends to a number of systems an impaired function of which mayhave the most serious consequences for the body.

Considering the importance of preserving zinc homeostasis to ensure aproper physiological status of all body tissues, adipose tissue inparticular, it appears that the obtained data on the positive effect ofZn-64 stable isotope in aspartate form in obesity are partly associatedwith the restoration of zinc levels, reduced during the pathogenesis ofobesity.

According to the accumulated data on the physiological role of zinc, anincrease in its levels may cause a cascade of biochemical shifts, which,ultimately, determine its overall positive effect.

There are several potential mechanisms for the implementation ofmodulating effects of Zn-64 stable isotope in aspartate form. First ofall, it is the enhancement of synthesis of zinc-dependent enzymes andtranscription factors. Increasing the number of key antioxidant enzymesdue to availability of zinc—a necessary structural component of theseenzymes—against the background of intensification of free radicalprocesses and the progression of oxidative stress will help to maintainan appropriate antioxidant reserve. An important fact is the ability ofzinc to induce the synthesis of a number of antioxidants,anti-inflammatory cytokines and factors that are actively involved inthe regulation of cellular signaling cascades and are involved in theregulation of basic processes. Thus, according to the literature data,the levels of zinc-alpha-glycoprotein (ZAG), which contributes to adecrease in fat deposits by stimulating lipolysis in adipocytes, issignificantly reduced in obesity. In addition, the process ofdifferentiation of adipocytes, brown adipose tissue in particular, isstrictly determined and controlled by a group of transcription factors,many of which contain zinc.

Thus, the results of the comprehensive analysis of the effects of Zn-64stable isotope in aspartate form on the pathogenesis of obesity showthis trace element as a promising additive to be used in the developmentof biologically active compounds not only for the treatment ofpathologies accompanied by a chronic inflammatory process, systemicdepletion of the antioxidant reserve and disturbances in the function ofthe serotonergic system but also for the prevention of diseasesassociated with systemic metabolic disorders.

Conclusions

For the first time, a comprehensive study into the effects of Zn-64stable isotope in aspartate form on the key pathogenetic links of thedevelopment and progression of obesity in diet-induced obesity modelswas conducted. The obtained results provide a factual basis foradvisability of the use of Zn-64 stable isotope in aspartate form as anauxiliary therapeutic agent in the treatment of overweight and obesepatients.

It has been shown that the administration of Zn-64 stable isotope inaspartate form to animals that were maintained on a high-fat diet isaccompanied by a decrease in their body mass index and helps to reduceweight and the amount of food consumed compared to the untreated animalmodels of obesity.

It has been found that Zn-64 stable isotope in aspartate form has apositive effect on the lipid metabolism in the body of animals that werefed a high-fat diet.

It has been found that Zn-64 stable isotope in aspartate form has apositive effect on the morphofunctional properties of the pancreas andliver of animals that were fed a high-fat compared to the untreatedanimal models of obesity.

It has been found that Zn-64 stable isotope in aspartate formadministered to animals fed a high-fat diet has a modulating effect onthe activity of key enzymes involved in serotonin metabolism, whichhelps to restore the levels of central and peripheral serotonin comparedto the values in the untreated animal models of obesity.

It has been shown that the administration of Zn-64 stable isotope inaspartate form contributes to the normalization ofprooxidant-antioxidant homeostasis in animals that were fed a high-fatdiet due to a decrease in the intensity of free radical processes(decrease in the levels of lipid peroxidation products and proteinoxidative modification) against the background of activation ofantioxidant defense via increased activity of antioxidant enzymes(superoxide dismutase and catalase).

The ability of Zn-64 stable isotope in aspartate form to influence acytokine profile in the serum and adipose tissue of animals maintainedon a high-fat diet, namely to reduce the levels of pro-inflammatorycytokines (IL-1, IL-6, IL-12, IFN-γ) against the background of a slightincrease in the levels of anti-inflammatory cytokines (IL-4, IL-10, TGF)has been shown, which generally indicates a decrease in the intensity ofsystemic inflammation.

It has been found that Zn-64 stable isotope in aspartate form has novisible effects on the levels of resistin in adipose tissue and bloodserum of animals having obesity, as well as on the serum levels ofghrelin.

It has been found that the administration of Zn-64 stable isotope inaspartate form causes redistribution of divalent metal ions (zinc,copper, manganese) between muscles, kidneys and liver in animal modelsof obesity and contributes to restoration of their physiological levels.

Example 7 Effects of Experimental Drugs on Fluctuations of InsulinLevels in the Blood of Laboratory Animals (Rats) by IntraperitonealAdministration

The first group consisted of animals that were injected with saline(body weight≈180-220 grams).

Number of animals: 8 (4 animals per group); 2 points of sampling—on thesecond day after the drug administration and on the 7th day after thelast injection of saline solution (4×2=8).

The second group consisted of animals that were injected with zincacetate with natural distribution of isotopes (body weight≈180-220grams). The current dose was calculated by zinc. Each animal wasinjected with 3750 μg of zinc per 1 kg of animal body weight. Acomparison group for evaluating the effects of zinc on experimentalanimals. Zinc acetate is a standard zinc compound used in most animalexperiments and diabetes experiments.

Number of animals: 8 (4 animals per group); 2 points of sampling—on thesecond day after the drug administration and on the 7th day after thelast injection of saline solution (4×2=8).

The third group consisted of animals that were injected with zincisotope in the form of aspartate (Zn⁶⁴) (body weight≈180-220 grams). Thecurrent dose was calculated by zinc. Each animal was injected with 3750μg of zinc per 1 kg of animal body weight.

Number of animals: 8 (4 animals per group); 2 points of sampling—on thesecond day after the drug administration and on the 7th day after thelast injection of saline solution (4×2=8).

The Scheme of Experiment:

Animals are placed in cages, 4 per cage, with free access to food/water.3 days after the animals have been in cages, they are injected with theexperimental drug—natural zinc or isotopically light zinc.Administration is performed every other day, in the amount of 7injections per animal (intraperitoneal method of drug administration).The dose of the drug injected to each animal was calculated on the basisof the ratio: 3750 μg of zinc per 1 kg of animal body weight. After thelast injection, a half of the animals are maintained on hunger (for 12hours) with free access to water. Upon expiration of this time period,the animals are taken out of the experiment. The second half of theanimals continues to have free access to food/water for a further 7days. On day 6 after the last injection, the animals are maintained onhunger (for 12 hours) with free access to water. Upon expiration of thistime period, the animals are taken out of the experiment.

Research Materials

Compliance with Animal Welfare Regulations that Govern Animal ResearchActivities

International recommendations for conducting biomedical research usinganimals in accordance with the General Principles of Working withAnimals, approved by the First National Congress on Bioethics (Kiev,Ukraine, 2001) and agreed with the provisions of the “EuropeanConvention for the Protection of vertebrates that are used forexperimental and other scientific purposes” (Strasbourg, France, 1986)were followed while working with laboratory animals. Experimental workwith rats was carried out in the vivarium of the Taras ShevchenkoNational University of Kyiv. Studies with animals were regulated by therules of experimental work with experimental animals, which wereapproved by the Scientific Council of this institution, which, in turn,were coordinated with the current legislation of Ukraine, adopted atthat time.

Conditions for Research Performance in Rats

Studies were performed on white rats aged from 2 to 3 months andweighing 120-300 g. Experimental animals were kept on a standardvivarium diet with free access to water. During the experiments, theanimals were kept at room temperature 19-24 ° C., humidity not more than50%, in a natural day-night light mode in plastic cages. Before theexperiments, animals were acclimatized in the research room for 7 days.

Preparation of Blood Serum of Rats

The rat serum was prepared from whole blood. To remove concomitantproteins and fibrinogen, the whole blood was left undisturbed at 37° C.for 30 minutes after which the samples were centrifuged at 2500 g for 15min. The resulting supernatant (serum) was immediately separated fromblood corpuscles and frozen at −20° C. for further analyses.

Preparation of Kidney and Liver Homogenates

The total kidney, liver and muscle homogenates were prepared as follows.Organ excision and homogenization were carried out at a temperature of1-4° C. Homogenization of tissues was carried out in 50 mM Tris-HClbuffer (pH 7.4) which contained 140 mM NaCl, 1 mM EDTA. The volume ofthe used buffer in ml was 5 times larger than the mass of isolatedorgans in grams. The isolated liver was perfused with chilled saline(0.9% NaCl) via the portal vein using a syringe. The minced liver wastransferred to a homogenizer with a finely ground Teflon pestle andhomogenized in chilled buffer. An isolated pair of kidneys was perfusedwith chilled saline, released from adipose tissue and minced withscissors. The minced tissue was transferred to a homogenizer with afinely ground Teflon pestle and homogenized in chilled buffer. The totalkidney and liver homogenates were centrifuged at 600 g for 15 minutes.The liquid was decanted after the mince settled down and was centrifugedagain at 15,000 g for 15 minutes. These two procedures allowed us to getrid of nuclear and mitochondrial debris. Aliquots of the preparedhomogenates were frozen in nitrogen (Rybalchenko V. K., Koganov M. M.Structure and function of membrane 1988.-312 pp).

1. Method for Determination of Insulin Level in Animal Serum:

Enzyme-Linked Immunosorbent Assay

(Insulin levels in blood serum of rats were determined using enzymeimmunoassay based on the common method used for soluble proteins. It wascarried out in 96-well microplates with sorption capacity for solubleproteins [Crowther J. R. The ELISA Guidebook/J. R. Crowther.—Totowa,N.J.: Humana Press Inc., 2001.-P. 436].

The serum was prepared as a 1 to 10 dilution with 50 mM Tris-HCl buffer(pH 7.4) containing 150 mM NaCl. Samples in volume of 100 μl wereincubated in microplate wells at 4° C. overnight. After incubation, thewells were washed with the buffer comprising 50 mM Tris-HCl buffer (pH7.4) with 150 mM NaCl and 0.05% Tween 20 to remove the unbound material.Non-specific binding sites were blocked with a 5% fat-free milk blockingsolution and incubated for 1 hour at 37° C. After washing, the separatewells of the microplate were loaded with primary rabbit anti-insulinantibodies and incubated for 1 hour at 37° C. After incubation, themicroplate wells were washed and loaded with appropriate secondaryantibodies conjugated to horseradish peroxidase and incubated foranother 1 hour at 37° C. The binding of secondary antibodies wasvisualized by adding 100 μl of OPD solution to each well at aconcentration of 0.4 mg/ml prepared in citrate buffer (pH 5.0)containing 0.013% H₂O₂. The optical density was measured at 492 nm. Theinsulin concentrations were calculated using the calibration curvegenerated under the given conditions and human insulin of knownconcentration.)

Determination of Superoxide Dismutase Activity in the Rat Liver andSerum

A method which is based on the ability of the enzyme to inhibit theprocess of adrenaline auto-oxidation was chosen to measure thesuperoxide dismutase (SOD, EC 1.15.1.1) activity is used.

10 μl aliquots of the test samples (liver and kidney homogenates) wereplaced into microplate wells. 200 μl of 0.2 M bicarbonate buffer (pH10.65) was added to each well. The reaction was started by adding 10 μlof a 0.1% solution of adrenaline to each well. No source of enzymes wasadded to the blank sample. The optical density was measured using amicroplate reader at a wavelength of 347 nm at the 4th and 8th minuteafter adrenaline was added.

A classical method of measuring enzymatic activity based on theaccumulated quantity of the product or decrease in the amount ofsubstrate is not applicable to SOD because it is impossible to constructa calibration curve for the reaction product which is the oxidizedproduct of adrenaline. The activity of this enzyme was expressed inconventional units/min*mg of protein which were calculated using thefollowing formula:

${A = \frac{X*50}{Y*4*100*a}},$

where X is the optical density of blank samples without test sampleequal to the difference between the optical density of the blank sampleat minute 8 and optical density of the same at minute 4; Y is theoptical density of blank samples with the test sample equal to thedifference between the optical density of the blank sample at minute 8and optical density of the same at minute 4; a is an amount of proteinin the blank sample, mg; 4 is incubation period between thedetermination of extinction, 4 min; 50/100 is conversion intoconventional units (Sirota T. V. Novel approach to the study ofadrenaline auto-oxidation and its use for the measurements of superoxidedismutase activity//Vopr Med Khim-1999.-45 (3).-P. 263-272).

Determination of Glucose Tolerance

Animals that had access only to water 16 hours before the start of theexperiment were used in the glucose tolerance test. Rats wereanesthetized with intraperitoneal injection of sodium thiopental at adose of 40 mg/kg. 5 animals were used within the same experimentalgroup. Basal levels of glycaemia were determined in rats, after whichthe animals were fed 2 ml of aqueous glucose solution at a dose of 3g/kg. The concentration of glucose was determined 60 minutes after thestart of the experiment. Blood collection was performed via the tailvein (Gorbulinska O. V. Sugar-lowering effects of water extracts ofyakon (Smallanthus sonchifolius poepp. & endl.)/O. V. Gorbulinska, M. R.Khokhla, L. T. Mishenko et. al.//Biological Studios—2014.-8 (2).-P.57-64.) Results see FIG. 16A-FIG. 16E.

According to all the data analyzed, there are no reliable differencesbetween the control groups (rats injected with saline) and the groupswhere rats were administered zinc. This may be due to two factors—eitherzinc solution was administered incorrectly (intraperitoneally instead oforally) or, since intact rats were used, they may have compensatorymechanisms which, in the event of an increase in the amount of zinc inthe body, do not allow the shift of the balance of the insular systemtowards hyperproduction of insulin, as this may result in hypoglycemiawith all that it entails.

At the same time, a significant increase in the body weight of rats isobserved in the group that was administered an isotope of zinc. This isa positive result, since people with type 1 diabetes often have adecrease in body weight. The obtained data may indicate that the isotopeof zinc may have a positive effect on the dynamics of weight gain.

Analysis of insulin levels in the serum of the experimental groups ofanimals showed the following results: no significant changes in theinsulin levels during the entire experiment were noted in the controlgroup of animals. In the group of animals injected with zinc acetate, asignificant increase is observed in the insulin levels on the first dayafter the drug withdrawal, and then a drop in the insulin levels to thevalue of those of intact/control animals. A reverse situation isobserved in the group which was given injections of zinc isotope. On thefirst day after the drug withdrawal, the insulin levels were slightlyhigher than in the group of intact animals, and at day 7 after thewithdrawal a significant increase in the insulin levels in this group ofanimals was observed. FIG. 17.

The results show that zinc isotope, as compared with zinc acetate,either has a more prolonged effect on insulin levels in blood serum oris more slowly released from the sites of deposition, and this alsocauses a slower effect of increasing insulin levels.

Analysis of the superoxide dismutase activity showed no significantchanges in its activity in serum in all experimental groups of animals.This may be due either to the lack of effect of the injected zincpreparations on the activity of this enzyme or (which is most likely) toincorrect administration of zinc preparations. FIG. 18A-FIG. 18B.

Microscopic study of islets of Langerhans has shown a positive dynamicsafter administration of zinc isotope—a significant increase in the isletarea compared to the control group and the group injected with zincacetate. FIG. 19.

The results obtained correlate with the results from the analysis ofinsulin levels in serum of the laboratory animals. Microscopic resultsindicate that the administration of zinc preparations, zinc isotope inparticular, leads to an increase in the area of pancreatic islets, whichin its turn may indicate the possibility of increasing insulinproduction by these islets. This is a move in the right direction, sincethe development of type I diabetes is associated with a significantinsulin deficiency due to problems with its synthesis by these islets.FIG. 20A-FIG. 20F.

TABLE 21 Glucose tolerance test (night on hunger, glucose at a dose of 3g/kg of body weight in a volume of 2 ml) 0 min 60 min Body (1 h after (1h after weight, effector glucose g administration) administration)Control (2 ml saline) 1 190 4.7 7.9 2 196 5.8 8 3 217 5.1 8 4 206 5.48.2 5 190 4.8 7.6 Zn isotope (dose: 5 mg/kg in a volume of 2 ml) 1 1954.5 6 2 178 4.8 6.3 3 187 4.5 6.5 4 186 4.7 6.6 5 184 4.8 6.5

A glucose tolerance test was performed to determine a potential effectof zinc isotope on the dynamics of glucose concentration in the bloodflow. Experimental procedure: laboratory animals are given intragastricinjections of either normal saline solution or zinc in saline solution(5 mg/kg) in a volume of 2 ml. After that, 60 minutes after theaforementioned injections, the basal level of glucose is measured and aglucose solution is injected in an amount of 3 g/kg in a volume of 2 mlusing the same route of administration. The blood glucose levels aremeasured again 60 minutes after injecting the glucose solution. Thedifference in the drop in glucose levels indicates a positive effect ofthe injected effector on the dynamics of glucose levels in thebloodstream.

The glucose tolerance test has shown a drop in glucose concentrationafter administering zinc isotope as compared to the control group. Thismay indicate the influence of administration of zinc isotope on theinsulin levels in the bloodstream, which in its turn results intriggering mechanisms associated with clearing the blood from glucose.Given that insulin is a zinc-dependent protein, it can be assumed thatthe administration of zinc causes either an increase in the activity ofthis protein relative to its receptor in tissues or an increase in theamount of this hormone in the bloodstream.

Analysis of accumulation of metals in the liver tissues (zinc, manganeseand copper) has shown that only zinc level significantly increases inboth groups of animals that received zinc preparations both at day 1after the drug withdrawal and at day 7 after the withdrawal. Thisindicates that zinc administered to the animals is accumulated and itsremoval does not increase. All other analyzed metals are within thelimits of their concentrations observed in the control group of animals.FIG. 21A-FIG. 21C.

Analysis of accumulation of metals in the kidney tissues (zinc,manganese and copper) has shown that only zinc level significantlyincreases in both groups of animals that received zinc preparations bothat day 1 after the drug withdrawal and at day 7 after the withdrawal.This indicates that zinc administered to the animals is accumulated andits removal does not increase. All other analyzed metals are within thelimits of their concentrations observed in the control group of animals.FIG. 22A-FIG. 22C. FIG. 23A-FIG. 23F.

Research Report on the Potential Effects of Test Substance (Zn⁶⁴Aspartate) on Type I Diabetes in Experimental Animals (Rats)

Oral Administration

The first group—Control. Weight of animals≈140-150 grams (initial bodyweight of animal).

Number of animals: 5 (males). All 5 animals were group-housed in onecage and provided ad libitum access to food and water. At the start ofthe experiment, each animal was given a single injection of 10 mMcitrate buffer (pH 4.5) intraperitoneally. 24 hours later, 7 doses ofthe test substance were administered to the animals orally. Frequency ofadministration—every other day. Dose of administration—800 μg of zincper animal.

After the last administration, the animals were fasted for 12 hours withad libitum access to water. Upon expiration of this time period, theanimals were taken out of the experiment

The second group—Diabetes. Weight of animals≈140-150 grams (initial bodyweight of animal).

Number of animals: 10 (males). Animals were housed in cages, 4 per cage,and provided ad libitum access to food and water. To induce diabetes inanimals, each animal was given a single injection of streptozotocinsolution at a dose of 6 mg per 100 g of animal weight dissolved in 10 mMcitrate buffer (pH 4.5) intraperitoneally. Dose of administration—800 μgof zinc per animal. The fasted animals were tested for sufficient levelsof glycemia two days after induction of diabetes. Rats with bloodglucose concentrations within the range >20 mmol/l were used in theexperiments.

The third group—Diabetes+zinc: Weight of animals≈140-150 grams (initialbody weight of animal).

Number of animals: 10 (males). Animals were housed in cages, 4 per cage,and provided ad libitum access to food and water. To induce diabetes inanimals, each animal was given a single injection of streptozotocinsolution at a dose of 6 mg per 100 g of animal weight dissolved in 10 mMcitrate buffer (pH 4.5) intraperitoneally. The fasted animals weretested for sufficient levels of glycemia two days after induction ofdiabetes. Rats with blood glucose concentrations within the range >20mmol/l were used in the experiments.

24 hours later, 7 doses of the test substance were administered to theanimals orally. Frequency of administration—every other day.

Dose of administration—800 μg of zinc per animal.

Research Materials

Compliance with Animal Welfare Regulations that Govern Animal ResearchActivities

The laboratory animals used in the experiments were maintained incompliance with international standards and recommendations on clinicaland biological research involving animals and in compliance with BasicPrinciples of Humane Animal Handling approved by the 1^(st) NationalCongress in Bioethics (Kyiv, Ukraine, 2001), which are in line with thestandards of the European Convention for the Protection of VertebrateAnimals used for Experimental and other Scientific Purposes (Strasbourg,Mar. 18, 1986). All experiments involving animals were performed in thevivarium of Taras Shevchenko National University of Kyiv according toanimal testing regulations developed in compliance with applicable lawsof Ukraine and approved by the Academic Board of the said institution.

Conditions of Maintenance of Laboratory Animals

White rats 2 to 3 months old weighing 120-130 g were used in theexperiments. The laboratory animals were fed standard vivarium diet andhad free access to water. During the experiments, the animals werehoused in plastic cages and environmental controls were set to maintainconditions of 19-24° C. and 50% relative humidity with a 12-h light-darkcycle. All animals were allowed to acclimatize to their environment for7 days before the onset of the experiments.

Determination of Glucose Concentration in Serum of Rats

The glucose concentration in whole blood was measured by the glucoseoxidase method using a GlucoDr Auto AGM-4000 blood glucose meter(Allmedicus Co., Ltd., Korea). All procedures were performed accordingto the manufacturer's instructions

Induction of Type I Experimental Diabetes in Rats

Type I experimental diabetes was induced by a single intraperitonealinjection of streptozotocin solution at a dose of 6 mg per 100 g ofanimal weight dissolved in 10 mM citrate buffer (pH 4.5). The rats ofthe control group were administered 10 mM citrate buffer (pH 4.5) by theaforementioned method. The whole blood glucose concentrations weremeasured two days after induction of diabetes in rats. Animals wereconsidered diabetic at blood glucose level reaching 22-32 mmol/L (M.Zafar, S. Naqvi//Int. J. Morphol.-2010.-Vol. 28, No 1.-P. 135-142.).

Preparation of Blood Serum of Rats

The rat serum was prepared from whole blood. To remove concomitantproteins and fibrinogen, the whole blood was left undisturbed at 37° C.for 30 minutes after which the samples were centrifuged at 2500 g for 15min. The resulting supernatant (serum) was immediately separated fromblood corpuscles and frozen at −20° C. for further analyses.

Determination of Glycated Hemoglobin in the Blood of Rats

The levels of glycated hemoglobin in whole blood of rats was measuredspectrophotometrically in accordance with the established procedure. Theassay was performed using a standard assay kit manufactured by Lachema(Czech Republic).

The method for glycated hemoglobin measurement is based on the fact thatthe stable form of glycohemoglobin (HbA1c) contains 1-deoxy-1-(N-valyl)fructose, which is dehydrated with phosphoric acid to form a coloredcomplex with an absorption spectrum at 433 nm. Neither the labile formof glycohemoglobin nor fetal hemoglobin interferes with thedetermination of glycated hemoglobin.

Total hemoglobin was measured spectrophotometrically. 20 μl of wholeblood was mixed with 5 ml of the transforming solution. The absorbancewas read at a wavelength of 540 nm against the transforming solution.The total hemoglobin fraction was calculated according to themanufacturer's recommendations using the following formula:

${Hb} = \frac{A*367\text{,}7}{4\text{,}92}$

where Hb is total hemoglobin, A is optical density of the test sample.The amount of total hemoglobin is expressed in g/L.

Hemolysate was prepared by adding an anticoagulant, 3.8% solution of Nacitrate diluted at 1:10, to freshly collected blood. 1 ml of stabilizedblood was collected and centrifuged at 1000 g for 10 min to removeplasma. 3 ml of saline was added to the obtained erythrocyte sediment,the mixture was gently stirred and centrifuged again, as describedabove. 3 ml of distilled water was added to the sediment and thewell-mixed mixture was allowed to stand at room temperature for 10minutes. After another centrifugation, 1.5 ml of supernatant(hemolysate) was separated and mixed with 0.25 ml of 85% phosphate acidsolution. The test tubes were closed with rubber stoppers and heated ina boiling water bath for 30 minutes. At dehydration, the tubes werecooled in running water for 10 minutes. 0.5 ml of a 2.45 M solution oftrichloroacetic acid was added to each tube. The contents of the tubeswere shaken and centrifuged at 1000 g for 20 minutes. To 1 ml aliquot ofsupernatant pipetted into another set of dry tubes, 2.5 μM ofthiobarbituric acid solution was added. The contents of the tubes werethoroughly mixed and incubated at 37° C. for 40 min. The samemanipulations were carried out for control samples, water was added toC1 instead of acid, and acid and a mixture of hemolysates from differentsamples were added to C2. The optical density of the samples wasmeasured on a spectrophotometer at a wavelength of 443 nm againstdistilled water.

Concentration of glycohemoglobin was calculated using the followingformula:

${{{HbA}\; 1\; c} = \frac{A_{1} - \left( {A_{2} - A_{3}} \right)}{{Hb}*K}},$

where A₁ is the optical density of the test sample, A₂ is the opticaldensity of the control sample for reagents, A₃ is the optical density ofthe positive control sample, K is the tangent of an angle calculated inaccordance with the fructose calibration curve, Hb is the totalhemoglobin content.

Concentration of glycohemoglobin was expressed as μmols of fructose perg of hemoglobin (Glycated hemoglobin/Assay kit//Pliva-lachemadiagnostica.-2008.-10003258.).

Determination of Insulin Levels in Rat Serum

Insulin levels in blood serum of rats were determined using enzymeimmunoassay based on the common method used for soluble proteins. It wascarried out in 96-well microplates with sorption capacity for solubleproteins.

The serum was prepared as a 1 to 10 dilution with 50 mM Tris-HCl buffer(pH 7.4) containing 150 mM NaCl. Samples in volume of 100 μl wereincubated in microplate wells at 4° C. overnight. After incubation, thewells were washed with the buffer comprising 50 mM Tris-HCl buffer (pH7.4) with 150 mM NaCl and 0.05% Tween 20 to remove the unbound material.Non-specific binding sites were blocked with a 5% fat-free milk blockingsolution and incubated for 1 hour at 37° C. After washing, the separatewells of the microplate were loaded with primary rabbit anti-insulinantibodies and incubated for 1 hour at 37° C. After incubation, themicroplate wells were washed and loaded with appropriate secondaryantibodies conjugated to horseradish peroxidase and incubated foranother 1 hour at 37° C. The binding of secondary antibodies wasvisualized by adding 100 μl of OPD solution to each well at aconcentration of 0.4 mg/ml prepared in citrate buffer (pH 5.0)containing 0.013% H₂O₂. The peroxidase reaction was stopped after 10 minby adding 100 μl of 1 M H₂SO₄. The optical density was measured at 492nm. Concentrations of insulin, cytokines and IgG were expressed inrelative units related to the total protein concentrations in serumdetermined using the Bradford protein assay.

Determination of Protein Concentrations

The protein concentrations were measured using the Bradford proteinassay, which is based on an absorbance shift of the dye CoomassieBrilliant Blue G-250.

To measure the protein concentration, 10 μl of 30% NaOH, 70 μl ofdistilled water, and 2 ml of Bradford reagent were added to each sample.To prepare 100 ml of Bradford reagent, 6 ml of stock solution, 3 ml of95% ethanol, 6 ml of 88% H₃PO₄ and 35 mg of Coomassie Brilliant Blue dyewere mixed and the resulting mixture was adjusted to the volume of 100ml with distilled water. The stock solution contained 10 ml of 95%ethanol, 20 ml of 88% H₃PO₄ and 35 mg of Coomassie Brilliant Blue.

The absorbance, which was visible in 2-5 min, was then measuredspectrophotometrically at 595 nm against a control sample that contained20 μl of distilled water instead of biomaterial. The proteinconcentration in each test sample was determined using a calibrationcurve and expressed in mg/ml.

Statistical Processing of the Results

Statistical processing of the obtained results was carried out using themethods of variation statistics and correlation analysis using OriginPro 7.0 and SPSS 16 software. Key statistical values were obtained bycalculations of the mean (M) and the standard error of the mean (m). Thedifference between variables was evaluated using a parametricstatistical technique (ANOVA). Student's t-test was used to evaluate thestatistical significance of differences between two samples. Thedifference was considered statistically significant when p<0.05.

Results

The study examining the potential effects of Zn⁶⁴ Aspartate on thedevelopment of type I diabetes in the experimental group of rats showedthe following results (Table 22). It was demonstrated that the mainparameters that characterize the development of type I diabetes, namelyconcentrations of glucose, glycated hemoglobin and insulin, improvedafter administration of the test substance. A significant decrease inglucose levels (by 26%) and glycated hemoglobin (by 30%) was registered.It was also found, that the serum insulin levels increased by 13%. Theobtained results may indicate that this test substance has a positiveeffect on the course of type I diabetes and may be used as aprophylactic agent to reduce the toxic effects of increased glucoselevels in the bloodstream during the development of this pathology.

TABLE 22 Values confirming the diagnosis of type I diabetes instreptozotocin-induced type I diabetes rat models Insulin, Glucose,Glycated hemoglobin, RU/mg mM μmol fructose/g Hb protein Control (n = 5)4.9 ± 0.1 0.34 ± 0.03 23 ± 1 Diabetes (n = 10)  27 ± 3* 0.72 ± 0.1* 15 ±3* Diabetes + ⁶⁴Zn  20 ± 3*^(#) 0.51 ± 0.05*^(#) 17 ± 2* Aspartate (n =10) *p < 0.05 versus the control group #p < 0.05 versus type I diabetesgroup

Cytokines are endogenous, biologically active polypeptide mediatorsrepresented by a large heterogeneous group of low-molecular,non-specific antigens, and glycoproteins that are produced in responseto an external extracellular stimulus and are involved in formation andregulation of specific immune responses in the body through theinteraction between non-specific protective reactions and specificimmunity. Pro-inflammatory cytokines, such as IL-1, IL-6, IL-8, IL-12,TNF-α, IFN-γ, take part in the launch of specific immune responses,whereas anti-inflammatory cytokines (IL-4, IL-10, IL-13, TGF) areinvolved in the development of anti-inflammatory reactions and inhibitthe synthesis of pro-inflammatory interleukins.

TABLE 23 Relative levels of pro-inflammatory cytokines IL-1β, IL-12 andIFN-γ in the serum of experimental animals (M ± m, n = 10) IL-1β IL-12IFN-γ Control 19.08 ± 0.71 8.25 ± 1.03 16.22 ± 0.35 Diabetes 26.16 ±0.51* 11.4 ± 1.85* 18.56 ± 0.63* Diabetes + zinc 22.27 ± 0.92*# 8.83 ±1.65# 15.24 ± 0.28*# *p < 0.05 versus the control group #p < 0.05 versustype I diabetes group

One of the controlling mechanisms for the levels and, accordingly, thebiological effects of pro-inflammatory cytokines is implemented by agroup of anti-inflammatory cytokines that includes IL-4, IL-10, IL-43,TGFβ. Cytokine imbalance is not only the basis for the occurrence ofinflammatory processes, but it also determines the further form of theimmune response, in particular whether it will be a predominantlycellular or humoral immune response. These cytokines are able to inhibitthe synthesis of pro-inflammatory cytokines by affecting transcriptionof specific genes in producer cells, induce the synthesis of interleukin1 receptor antagonists, enhance the production of soluble receptors andreduce the density of pro-inflammatory receptors on cells. Thus, IL-4

IL-10 inhibit production of PGE2, super and nitroxide radicals, andblock the formation of IL-1, IL-6, IL-8, TNF, inhibit the synthesis ofIL-2, IFN-γ in lymphocytes.

TABLE 24 Relative levels of anti-inflammatory cytokines IL-4, IL-10_(T)a TGF-β in the serum of experimental animals (M ± m, n = 10) IL-4IL-10 TGF-β Control 2.41 ± 1.22  1.23 ± 1.05 3.22 ± 0.35 Diabetes 5.97 ±0.57*  8.83 ± 0.25* 5.56 ± 0.63* Diabetes + zinc 6.42 ± 0.61* 14.93 ±0.56*# 8.24 ± 0.28*# *p < 0.05 versus the control group #p < 0.05 versustype I diabetes group

The results of this study showed a certain positive effect of Zn⁶⁴Aspartate on the cytokine profile, which can be regarded as a factorthat normalizes an inflammatory process that occurs in the body duringthe development of type I diabetes.

Summarizing Results of the Study

The present study into the potential effects of the test substance onthe glucose metabolism and related processes have shown the prospects ofits use to normalize glucose metabolism in the body in patientsdeveloping type I diabetes. Presumably it can be used as an independentagent, as well as in combination with other drugs.

Intraperitoneal route of administration of the test substance is notsuitable as a potential regulator of glucose metabolism. Oraladministration showed a much better effect and led to a decrease in theglucose levels in the control animals.

The results show that zinc isotope, as compared with zinc acetate(natural zinc), either has a more prolonged effect on insulin levels inblood serum or is more slowly released from the sites of its deposition,and this also causes a slower effect of increasing insulin levels.

The results of microscopic examination indicate that the administrationof zinc preparations, zinc isotope in particular, leads to an increasein the area of pancreatic islets, which in its turn may indicate thepossibility of increasing insulin production by these islets. This is amove in the right direction, since the development of type 1 diabetes isassociated with a significant insulin deficiency due to problems withits synthesis by these islets. The results obtained correlate with theresults from the analysis of insulin levels in serum of the laboratoryanimals.

The glucose tolerance test showed a significant decrease in glucoselevels after administration of zinc isotope ⁶⁴Zn as compared to thecontrol group. This may indicate the influence of administered testsubstance on the insulin levels in the bloodstream, which in turnresults in triggering mechanisms associated with clearing glucose fromthe blood. Given that insulin is a zinc-dependent protein, it can beassumed that the administration of zinc causes either an increase in theactivity of this protein relative to its receptor in tissues or anincrease in the amount of this hormone in the bloodstream.

The results obtained using a type I diabetes model may indicate thatthis test substance has a positive effect on the course of type Idiabetes and may be used as a prophylactic agent to reduce the toxiceffects of increased glucose levels in the bloodstream during thedevelopment of this pathology.

The results of this study showed a certain positive effect of Zn⁶⁴Aspartate on the cytokine profile, which can be regarded as a factorthat normalizes an inflammatory process that occurs in the body duringthe development of type I diabetes.

Based on mathematical model of a stable system, mass-spectrometryexperimental data and analysis of the literature sources, the change ofamino acids chirality in proteins with subsequent violation of proteinsconformation and defects in DNA are the result of distortion in helixesbecause of stable isotopes substitution. There is a strong evidence tosuggest that the isotope composition of the same chemical elementsaffects chemical bonds formation in solenoidal/helical structures ofbiomolecules. Isotope induced changes in biomolecules conformationappear to represent the onset of pathologies. But the most importantfeature of such changes is their reversibility. Conformation ofbiomolecules can be corrected with the modulation of isotope ratios ofelements they are made from. Perfect conformation of proteins meansperfect healthy/youthful homeostasis. It allows for complex effect onpathological changes in cells, tissues, organs and organism with immune,endocrine and nervous systems all mobilized against degenerativediseases. Isotope selective therapy can be the next step after treatmentstrategy based on molecular signatures.

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of theappended claims. Thus, while only certain features of the invention havebeen illustrated and described, many modifications and changes willoccur to those skilled in the art. It is therefore to be understood thatthe appended claims are intended to cover all such modifications andchanges as fall within the true spirit of the invention.

1. A method of preventing overweightness or obesity comprisingadministering to a subject in need thereof a prophylactically ortherapeutically effective amount composition comprising a ⁶⁴Zn_(e)compound or a salt thereof, wherein the ⁶⁴Zn_(e) compound or a saltthereof is at least 80% ⁶⁴Zn_(e).
 2. The method of claim 1, wherein thecomposition further comprises a diluent or an excipient.
 3. The methodof claim 2, wherein the diluent is deuterium-depleted water.
 4. Themethod of claim 1, wherein the ⁶⁴Zn_(e) compound or a salt thereof is atleast 95% ⁶⁴Zn_(e).
 5. The method of claim 1, wherein the ⁶⁴Zn_(e)compound or a salt thereof is at least 99% ⁶⁴Zn_(e).
 6. The method ofclaim 1, wherein the composition contains between 0.05 mg and 110 mg of⁶⁴Zn_(e).
 7. The method of claim 6, wherein the composition containsbetween 1 and 10 mg of ⁶⁴Zn_(e).
 8. The method of claim 1, wherein the⁶⁴Zn_(e) compound or a salt thereof is at least 90% ⁶⁴Zn_(e) and thecomposition is an aqueous solution in which ⁶⁴Zn_(e) is present at aconcentration of between 0.1 mg/ml and 10 mg/ml.
 9. The method of claim1, wherein ⁶⁴Zn_(e) is in a form of salt selected from the groupconsisting of zinc aspartate with 2 aspartic acid molecules, zincsulfate, and zinc citrate wherein the zinc aspartate has the structure


10. The method of claim 1, wherein the composition is administered byinjection.
 11. The method of claim 1, wherein the composition isadministered orally.
 12. A method of treating overweightness or obesitycomprising administering to a subject in need thereof a prophylacticallyor therapeutically effective amount composition comprising a ⁶⁴Zn_(e)compound or a salt thereof, wherein the ⁶⁴Zn_(e) compound or a saltthereof is at least 80% ⁶⁴Zn_(e), wherein ⁶⁴Zn_(e) is in a form of saltselected from the group consisting of zinc aspartate with 2 asparticacid molecules, zinc sulfate, and zinc citrate, wherein the zincaspartate has the structure